Transgenic Non-Human Vertebrate for the Expression of Class-Switched, Fully Human, Antibodies

ABSTRACT

The present invention relates to humanisation of antibodies in vivo. The invention provides non-human vertebrates, cells, populations and methods useful for humanising chimaeric antibodies in vivo. Using the present invention, it is possible straightforwardly and rapidly to obtain antigen-specific antibodies that are fully human (i.e., comprising human variable and constant regions) and have undergone recombination, junctional diversification, affinity maturation and isotype switching in vivo in a non-human vertebrate system. Furthermore, such antibodies are humanised (e.g., totally human)—and selected—totally in vivo, and as such the present invention harnesses in vivo filtering for expressibility, affinity and biophysical characteristics in the context of the desired human variable and constant region pairings. This avoids problems of down-grading antibody characteristics when humanising the constant region of chimaeric antibodies in vitro.

CROSS REFERENCE

This application is a Divisional application of U.S. application Ser.No. 16/353,870, filed on Mar. 14, 2019, which is a Continuation of U.S.application Ser. No. 14/498,685 filed Sep. 26, 2014, now U.S. Pat. No.10,251,377, issued Apr. 9, 2019, which is a Continuation ofPCT/GB2013/050683 filed Mar. 18, 2013, which claims priority to U.S.application Ser. No. 13/433,084 filed Mar. 28, 2012, U.S. applicationSer. No. 13/434,361 filed Mar. 29, 2012, GB Application No. 1207814.3filed May 4, 2012, GB Application No. 1208708.6 filed May 17, 2012 andGB Application No. 1216795.3 filed Sep. 20, 2012, the contents of all ofwhich are herein incorporated by reference in their entirety.

Humanisation of Affinity Matured Antibodies & Heavy Chains In Vivo

The present invention relates to the humanisation of antibodies andheavy chains in a way that produces useful human heavy chain repertoiresby harnessing endogenous control of junctional mutation and affinitymaturation in the context of desired constant regions in vivo innon-human vertebrate systems. To this end, the invention providesnon-human vertebrates, cells, populations and methods useful forhumanising chimaeric antibodies and heavy chains in vivo. Using thepresent invention it is possible straightforwardly and rapidly to obtainantigen-specific antibodies and heavy chains that are fully human (ie,comprising human variable and constant regions) and have undergonejunctional mutation and affinity maturation in vivo by harnessing amouse or other non-human vertebrate system. Furthermore, such antibodiesand heavy chains are humanised—and selected—totally in vivo, and as suchthe present invention harnesses in vivo filtering for expressibility(and provides possibly for aspects of affinity and biophysicalcharacteristics to be factored into in vivo selection) in the context ofthe desired human variable and constant region pairings. This is avoidsproblems of down-grading antibody characteristics when humanising theconstant region of chimaeric antibodies and heavy chains in vitro. Thus,the present invention allows the skilled person to select antibodies andheavy chains directly in the format that they will be used forsubsequent human therapeutic and prophylactic use.

BACKGROUND

The state of the art provides methods for producing antibodies in vitro(eg, using phage, ribosome or yeast display) or in vivo (eg, usingnon-human vertebrates (eg, mice and rats) and cells comprisingtransgenic immunoglobulin loci). Such in vivo systems (eg, Xenomouse™)have used completely human transgenic heavy chain loci which comprisehuman variable regions (human VH, D and JH gene segments) upstream ofhuman constant regions (eg, human mu upstream of human gamma constantgene segments). Subsequently, it has been discovered that the use oftotally human transgenic loci in such in vivo systems is detrimental andB-cell development is hampered, leading to relatively small B-cellcompartments and restricted utility for generating antibodies.Later-generation transgenic animals (eg, the Velocimouse™) have beencreated which have chimaeric heavy chain loci in which a human variableregion is upstream of endogenous (eg, mouse or rat) constant regions(ie, mouse mu constant region upstream of gamma constant region, ingermline configuration). This enables the harnessing of endogenouscontrol mechanisms for B-cell and antibody development, and as such theextent of problems of totally human transgenic loci are not seen.Methods of constructing transgenic vertebrates and use of these togenerate antibodies and nucleic acids thereof following antigenimmunisation are known in the art, eg, see U.S. Pat. No. 7,501,552(Medarex); U.S. Pat. No. 5,939,598 & U.S. Pat. No. 6,130,364 (Abgenix);WO02066630, WO2011163311 & WO2011163314 (Regeneron); WO2011004192 &WO2011158009 (Kymab Limited); WO2009076464, WO2009143472, EP1414858,WO2009013620A2, WO2010070263A1 & WO2010109165A2 (Harbour Antibodies);EP1399559 (Crescendo Biologics) and WO2010039900 (Ablexis), thedisclosures of which are explicitly incorporated herein including, butnot limited to, for the purpose of providing the skilled person withguidance of how to make non-human animals bearing transgenicimmunoglobulin loci and to inactivate endogenous loci expression.US2008/0196112A1 (Innate Pharma) discloses transgenic animals comprisinga single, predetermined human rearranged VDJ from a lead antibody,together with one or more human constant region genes in a locus. Thereare no repertoires of unrearranged V, D and J and recombination andjunctional diversity to produce a repertoire of VH domains and H chainsand antibodies is not addressed.

Whether selected and produced in vitro or in vivo, the art hasrecognised that the human therapeutic utility of chimaeric antibodies ishampered by their non-human constant domains, despite having humanvariable domains. This is due to the immunogenicity of the non-humanportions once the antibodies have been administered to human patients.The solution in the art to this issue has been to humanise chimaericantibodies in vitro using protein engineering whereby the non-humanconstant domains are replaced with corresponding human constant domains.While addressing the immunogenicity issue, however, such engineering invitro can downgrade the desirable characteristics of the resultantantibody. For example, antigen-binding affinity, antigen specificity,expressibility (eg, in cell lines such as CHO or HEK293 cells),half-life and/or biophysical characteristics (eg, melting temperature,solution state, resistance to aggregation etc) can be downgraded,thereby hampering development of the antibody as drugs for humantherapeutic or prophylactic use. It would be desirable to have means forselecting and humanising antibodies that addresses these shortcomings inthe art as well as providing repertoires of such humanised antibodies orheavy chains from which drug candidates can be selected.

SUMMARY OF THE INVENTION

The present invention provides a solution by providing for therecombination, humanisation and selection of antibodies entirely in vivoby harnessing the control mechanisms of non-human vertebrate systems. Tothis end, the invention provides non-human vertebrates, cells,populations and methods useful for humanising chimaeric antibodies toproduce fully human antibodies that have undergone variable region genesegment recombination, junctional mutation, somatic hypermutation andisotype switching in vivo and that are selected by an in vivo system.Using the present invention it is possible straightforwardly and rapidlyto obtain antigen-specific antibodies that are fully human (ie,comprising human variable and constant regions) and have undergonerecombination, affinity maturation in vivo. Furthermore, such antibodiesare humanised—and selected—totally in vivo, and as such the presentinvention harnesses in vivo filtering for expressibility (and affinityand biophysical characteristics factors) in the context of the desiredhuman variable and constant region pairings. This is avoids problems ofdown-grading antibody characteristics when humanising the constantregion of chimaeric antibodies in vitro.

Importantly, the present invention does this in a way that harnessesendogenous control of B-cell development and antibody production andselection. The invention achieves this by ensuring that these functionsare carried out in situ in a non-human vertebrate system in anendogenous IgH locus where heavy chain development and selection passesthrough an IgM stage that is matched with the non-human vertebratesystem. Thus, the invention adapts an endogenous IgH locus by targetedinsertion of unrearranged human variable region gene segments therein infunctional relationship upstream of an endogenous mu constant region(ie, the endogenous Cmu or an inserted exogenous Cmu of the same speciesof non-human vertebrate). Thus, endogenous control of recombination andIgM heavy chain development and selection can be harnessed to provide auseful pool of properly developed B-cells expressing chimaeric humanV/endogenous Cmu heavy chains. Provision of such a good repertoire ofcell-surface expressed IgM heavy chains is advantageous since itprovides the non-human system with a useful and diverse repertoire fromwhich to select and clonally mature B-cells for subsequent isotypeswitching to a human non-mu constant region. Thus, a useful repertoireof human non-mu (eg, gamma) heavy chains is provided that have undergonerigorous selection by the endogenous non-human system in vivo forantigen binding and expressibility. The skilled person is thus able toselect and isolate one or more desired heavy chains (or antibodiescomprising a heavy chain) that are specific to a target antigen, knowingthat these have been selected in a totally human format forcompatibility with in vivo systems. Thus, one or more biophysicalcharacteristics (eg, melting temperature, low aggregatability,expressibility etc as will be known by the skilled person) have alreadybeen usefully tuned, thereby making the selected heavy chain or antibodyuseful for human drug production.

Thus, the invention provides:—

A non-human vertebrate (eg, a rodent, a mouse, a rat or a rabbit) or anon-human vertebrate cell (eg, a rodent cell, a mouse cell, a rat cellor rabbit cell) whose genome comprises a gene locus for expression ofantibody heavy chains, the locus comprising

(a) an unrearranged human variable region comprising human variableregion gene segments for expression of a repertoire of human variabledomains;(b) an endogenous mu constant region for expression of IgM antibodyheavy chains comprising endogenous mu heavy chain constant domains andhuman variable domains; and(c) a humanised non-mu constant region downstream of the mu constantregion for expression of non-mu antibody heavy chains comprising humannon-mu constant domains and human variable domains;Wherein the unrearranged variable region is provided as a targetedinsertion of the human variable region gene segments upstream of theendogenous mu constant region in an endogenous IgH locus such that thevariable region gene segments are able to recombine for expression andselection in the context of an endogenous mu constant region.

In alternative embodiments of any configuration or aspect of theinvention, the mu constant region is not endogenous, but is of adifferent non-human vertebrate species (ie, different to the species ofvertebrate or cell). For example, the vertebrate or cell is a mouse andthe mu constant region is a non-mouse rodent (eg, rat) mu constantregion. For example, the vertebrate or cell is a rat and the mu constantregion is a non-rat rodent (eg, mouse) mu constant region. By selectingrelated species, for example, it is possible still for the endogenouscontrol to function in the context of the mu constant region. Thus, inthese embodiments, the vertebrate or cell is of a first non-humanvertebrate species and the mu constant region is of a second (ie,different) non-human vertebrate species.

In embodiments of any configuration or aspect of the invention, the muconstant region is of the same non-human species as the vertebrate orcell, but is of a different strain (eg, different mouse or rat strainwhere the vertebrate is a mouse or rat, or the cell is a mouse or ratcell).

The invention also provides embodiments wherein the IgH locus is providenot by targeted insertion, but by insertion of a transgene and randomintegration into the genome or integration in a location that is not theendogenous wild-type IgH locus. For example, an IgH locus comprisinghuman variable region and human non-mu constant region can be providedin a yeast artificial chromosome (YAC), PAC or other vector known in theart for accommodating large inserts. The vector can be introduced intoan ES cell (eg, mouse or rat ES cell) and the IgH locus can integrateinto the genome of the cell.

In embodiments of any configuration or aspect of the invention thehumanised non-mu constant region is totally human (ie, comprises onlyhuman constant domain genes).

Thus, the invention provides:—

A non-human vertebrate (eg, a rodent, a mouse, a rat or a rabbit) or anon-human vertebrate cell (eg, a rodent cell, a mouse cell, a rat cellor rabbit cell) whose genome comprises a gene locus for expression ofantibody heavy chains, the locus comprising

(a) an unrearranged human variable region comprising human variableregion gene segments for expression of a repertoire of human variabledomains;(b) an endogenous mu constant region for expression of IgM antibodyheavy chains comprising endogenous mu heavy chain constant domains andhuman variable domains; and(c) a humanised non-mu constant region downstream of the mu constantregion for expression of non-mu antibody heavy chains comprising humannon-mu constant domains and human variable domains;Wherein the unrearranged variable region is provided upstream of theendogenous mu constant region in the locus such that the variable regiongene segments are able to recombine for expression and selection in thecontext of an endogenous mu constant region.

The invention also provides:—

A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebratecell (eg, a mouse cell or a rat cell) whose genome comprises a genelocus for expression of antibody heavy chains, the locus comprising

(a) a human variable region comprising human variable region genesegments for expression of human variable domains;(b) an endogenous mu constant region for expression of IgM antibodyheavy chains comprising endogenous mu heavy chain constant domains andhuman variable domains; and(c) a humanised non-mu constant region downstream of the mu constantregion for expression of non-mu antibody heavy chains comprising humannon-mu constant domains and human variable domains.

Also the invention provides a non-human vertebrate (eg, a mouse or arat) or a non-human vertebrate cell whose genome comprises an antibodyheavy chain locus comprising (in 5′ to 3′ direction) a variable region,a first switch, an endogenous mu constant region, a second switch and ahuman non-mu (eg, gamma) constant region; wherein the heavy chain locusof each cell is capable of undergoing switching from IgM to the non-mu(eg, IgG) isotype.

By retaining an endogenous IgM stage, the invention retains the abilityto harness the endogenous antibody and B-cell development controlmechanisms of the non-human vertebrate or cell. This includes theability to harness endogenous terminal deoxynucleotidyl transferase(TdT) activity and junctional mutation as well as proper B-celldevelopment (including at early stages of development when IgM isexpressed) to yield good size B-cell compartments and good size andquality repertoires of recombined VDJ diversity from which tosubsequently select and mature isotype-switched heavy chains producedfrom the gene locus of the invention. Furthermore, this entailsswitching to a human constant region in resulting antibodies, which isbeneficial for producing drugs for human therapeutic use. Thus, forexample, a vertebrate of the invention can be immunised with apredetermined therapeutic target antigen (eg, a human, viral orbacterial antigen) and antibodies (eg, human IgG) or heavy chains (eg,human gamma-type heavy chains) can be isolated that (i) have undergonejunctional and somatic mutation, and thus affinity maturation in vivo inresponse to antigen challenge, (ii) are fully human, ie, have humanvariable and constant domains, and (iii) have been selected by a totallyin vivo system for expression and ability to bind antigen. Thus, theinvention employs in vivo systems to direct the skilled person to viablehuman antibodies and heavy chains that can be expressed well in vivo andwhich are specific for a predetermined antigen. The method is simple andavoids multi-step combinations of in vivo and in vitro production andengineering as per the prior art methods.

Moreover, having undergone affinity maturation in the vertebrate orcell, the selected antibodies have had their antigen binding affinitiestuned by nature—rather than by man. Thus, vertebrates and cells of theinvention provide good sources for fully human antigen-specificantibodies and heavy chains with therapeutically-amenable affinitieswithout the need to perform laborious in vitro affinity maturation (orrisk downgrading desirable characteristics due to in vitromanipulation).

Additionally, the present invention enables the skilled person to designvertebrates and cells to produce fully human antibodies of a specific,desired isotype in a predictable way. For example, the skilled person isable to design the heavy chain loci in a vertebrate of the invention sothat all gamma constant region gene segments are human ones. In thisway, the skilled person will know that all IgG-type antibodies or heavychains selected from the vertebrate will be fully human in their Hchains and will have been subjected to endogenous antibody development(eg, affinity maturation) and in vivo expression and selection. Going astep further, the skilled person could design a vertebrate in which justthe IgG1 isotype gene segments are human. Thus, the skilled person willknow from the outset that all IgG1 isotype antibodies will be fullyhuman and matured, expressed and selected in vivo. This is useful fortailoring antibody sub-types, such as IgG1 or IgG4, to particular humantherapeutic indications, as explained further below.

Thus, in an embodiment, the gene locus of the invention comprises onlyhuman constant regions, with the exception of the endogenous mu constantregion. This is useful for producing isotype-switched heavy chains inwhich the constant regions are predictably always human.

In an embodiment, the gene locus of the invention comprises only humanconstant region gene segments, with the exception of the constant regiongene segments of the endogenous mu constant region. Thus, usefully, theskilled person will reliably know at the outset that all switched heavychains and antibodies will bear human heavy chain constant domains.

In an embodiment, the gene locus of the invention comprises gammaconstant region gene segments and all of the gamma constant region genesegments are human. This is useful for producing isotype-switched gammaheavy chains in which the constant regions are predictably always human.

In an embodiment, the gene locus of the invention comprises gammaconstant region gene segments and all of the delta constant region genesegments are human. This is useful for producing isotype-switched deltaheavy chains in which the constant regions are predictably always human.

In an embodiment, the gene locus of the invention comprises epsilonconstant region gene segments and all of the delta constant region genesegments are human. This is useful for producing isotype-switchedepsilon heavy chains in which the constant regions are predictablyalways human.

In an embodiment, the gene locus of the invention comprises alphaconstant region gene segments and all of the delta constant region genesegments are human. This is useful for producing isotype-switched alphaheavy chains in which the constant regions are predictably always human.

The invention also enables the skilled person to tailor othercharacteristics of antibody constant regions to particular therapeuticor prophylactic uses. For example, one can include variant human non-muconstant region gene segments in the heavy chain locus. Thus, in oneembodiment, the variants may encode mutant human Fc regions that areeither activated (eg, for cell-killing applications such as oncology ortreatment of inflammatory conditions) or inactivated (eg, whenactivation due to cross-linking of cell-surface antigens by antibody isundesirable, eg, as seen when the target antigen is CD40 or CD40ligand).

In another configuration, the application recognises the desirability ofexploring repertoires of variant human constant domains of antibodieswhen paired and selected with human variable domains in vivo. Suchvariants are seen in different populations of humans (eg, encoded bydifferent variants of gamma constant region gene segments as seen indifferent ethnic human populations). Similarly, this concept can beapplied to synthetic variants of human constant domains andcorresponding human constant region gene segments (eg, variants thathave been mutated in different ways to inactivate Fc, or to activateFc). To this end, the invention provides:—

A population of vertebrates of the invention, wherein the populationcomprises

(i) a first vertebrate wherein the antibody heavy chain locus comprisesone or more first human non-mu gene segment(s) (eg, a human CH1, CH2 orCH3; or an Fc);(ii) and a second vertebrate the antibody heavy chain locus comprisesone or more second human non-mu gene segment(s) (eg, a human CH1, CH2 orCH3; or encoding an Fc); andwherein the first and second gene segments are the same type of constantregion gene segment(s) (eg, both CH1 gene segments or both encoding anFc) and the second gene segment(s) is a variant of the first genesegment(s).

In an embodiment, the immunoglobulin loci of the vertebrates of thepopulation differ only in the repertoire of said human constant regionsor constant region gene segments.

The invention also provides a method of isolating a human antibody, themethod comprising immunising a vertebrate or population of vertebratesaccording to any preceding claim with an antigen and isolating anantibody from said vertebrate or a vertebrate of the population, whereinthe isolated antibody specifically binds to the antigen and comprises afully human heavy chain of said non-mu isotype.

The invention also provides:—

A method of obtaining a humanised and affinity matured antigen-specificantibody heavy chain, the method comprising humanising the heavy chainin vivo in a non-human vertebrate (eg, a mouse or a rat) comprisingfunctional RAG and activation induced cytidine deaminase (AID) byimmunising the vertebrate with the antigen and obtaining recombinationof VH,D and JH gene segments in vivo, somatic hypermutation and isotypeswitching in a B-cell of the vertebrate from an endogenous mu isotype toa human non-mu isotype, wherein a repertoire of affinity maturedantigen-specific non-mu antibody heavy chains are produced and expressedby the vertebrate, the non-mu constant domains of the heavy chains beinghuman constant domains, the method further comprising isolating one ormore of said humanised heavy chains.

By using a non-human vertebrate ES cell (eg, an ES cell disclosedherein) the skilled person will be able to provide for the provision offunctional RAG and AID. These will be matched with the endogenous mu forgood harnessing of the endogenous control mechanisms.

A method of obtaining a humanised and affinity matured antigen-specificantibody heavy chain, the method comprising humanising the heavy chainin vivo in a non-human vertebrate (eg, a mouse or a rat) comprising afunctional activation induced cytidine deaminase (AID) by immunising thevertebrate with the antigen and obtaining somatic hypermutation andisotype switching in a B-cell of the vertebrate from an endogenous muisotype to a human non-mu isotype, wherein an affinity maturedantigen-specific antibody heavy chain is produced and expressed by thevertebrate, the non-mu constant domains of the heavy chain being humanconstant domains, the method further comprising isolating said humanisedheavy chain.

The invention also provides a pharmaceutical composition comprising theisolated antibody heavy chain obtained in the method of the invention ora copy or derivative thereof; optionally wherein the heavy chain, copyor derivative is provided by an antibody that specifically binds theantigen.

The invention also provides a method of treating or preventing a medicalcondition or disease in a human associated or caused by said antigen,the method comprising administering to the human the antigen-specificantibody or heavy chain obtained in the method described above.

The invention also provides the antigen-specific antibody or heavy chainobtained in the method of the invention for use in the treatment orprophylaxis of a medical condition or disease in a human associated orcaused by said antigen.

The invention also provides the use of the antigen-specific antibody orheavy chain obtained in the method of the invention in the manufactureof a medicament for use in the treatment or prophylaxis of a medicalcondition or disease in a human associated or caused by said antigen.

FIGURES

FIG. 1 is a schematic showing the arrangement of the constant regions inwild-type human and mouse genomes (Dev Comp Immunol. 2006;30(1-2):119-35; Schroeder H W Jr);

FIG. 2A-2D is a schematic showing the arrangement of gamma, delta,alpha, mu and epsilon isotype human constant regions as found in genomicheavy chain loci (see IMGT database; www.IMGT.org).

FIG. 3A-3D is a schematic showing the arrangement of gamma, delta,alpha, mu and epsilon isotype mouse constant regions as found in genomicheavy chain loci (see IMGT database; www.IMGT.org).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to humanisation of antibodies in vivo. Tothis end, the invention provides non-human vertebrates, cells,populations and methods useful for humanising chimaeric antibodies invivo. Using the present invention it is possible straightforwardly andrapidly to obtain antigen-specific antibodies that are fully human (ie,comprising human variable and constant regions) and have undergoneaffinity maturation in vivo. Furthermore, such antibodies arehumanised—and selected—totally in vivo, and as such the presentinvention harnesses in vivo filtering for expressibility (and factors inaffinity and biophysical characteristics) in the context of the desiredhuman variable and constant region pairings. This is avoids problems ofdown-grading antibody characteristics when humanising the constantregion of chimaeric antibodies in vitro.

To this end, the invention provides a non-human vertebrate (eg, a mouseor a rat) or a non-human vertebrate cell (eg, a mouse cell or a ratcell) whose genome comprises a gene locus for expression of antibodyheavy chains, the locus comprising

(a) a human variable region comprising human variable region genesegments for expression of human variable domains;(b) an endogenous mu constant region for expression of IgM antibodyheavy chains comprising endogenous mu heavy chain constant domains andhuman variable domains; and(c) a humanised non-mu constant region downstream of the mu constantregion for expression of non-mu antibody heavy chains (eg, followingimmunisation of the vertebrate with target antigen) comprising humannon-mu constant domains and human variable domains.

The locus is, in the preferred embodiment, a heavy chain locus of thegenome, ie, it is in the germline location of a heavy chain locus ofsaid type of vertebrate or cell. In this example, therefore, thehumanised locus of the invention can be constructed by inserting humanconstant region DNA into the endogenous (ie, non-human vertebrate) heavychain locus downstream of the endogenous mu constant region. In anotherembodiment, the locus of the invention is synthetically constructedelsewhere in the genome (ie, not in the endogenous heavy chain locuslocation) and may be on a different chromosome to that chromosomeharbouring the heavy chain locus in a wild-type vertebrate or cell. Forexample, the locus of the invention is constructed at the Rosa26 locus.

The gene locus of the invention comprises all of the elements requiredfor expression of heavy chains and isotype switching in the vertebrateor cell, such a human variable region comprising a plurality of of VHgene segments, a plurality of D gene segments and a plurality of JH genesegments upstream of (ie, 5′ of) a S-mu switch (eg, the endogenousS-mu), an endogenous mu constant region comprising endogenous muconstant gene segments (eg, CH1, CH2 and CH3), a second switch (eg, anendogenous switch; eg, a gamma switch) and a (ie, one or more) humannon-mu constant region. For example, the human non-mu constant region isa gamma-1 constant region comprising from CH2 to (and including) thepoly-A 3′ of M2 as shown in FIG. 3. Alternatively, the non-mu constantregion is a gamma-1 constant region comprising from CH1 to (andincluding) the poly-A 3′ of M2 as shown in FIG. 3. Alternatively, thenon-mu constant region is a gamma-1 constant region comprising from thehinge region (H) to (and including) the poly-A 3′ of M2 as shown in FIG.3. Optionally, the human constant region lacks a CH1 gene segment butincludes one or more other human constant gene segments (eg, CH2 andCH3); optionally wherein the constant region is a gamma constant region.This example is useful for producing heavy chain-only antibodies (H2antibodies) when the vertebrate or cell does not express functionallight chains (or expresses no light chains) after isotype switching tothe non-mu isotype.

Thus, the following examples of inserted DNA comprising one or morehuman non-mu constant regions are contemplated:—

Human Gamma Constant Regions

-   -   1. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising or consisting of the sequence from        CH1 to (and including) CH3, M1, M2 or the poly-A immediately 3′        of CH3 or M2;    -   2. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising or consisting of the sequence from a        hinge to (and including) CH3, M1, M2 or the poly-A immediately        3′ of CH3 or M2;    -   3. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising or consisting of the sequence from        CH2 to (and including) CH3, M1, M2 or the poly-A immediately 3′        of CH3 or M2;    -   4. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising or consisting of the sequence from        CH3 to (and including) M1, M2 or the poly-A immediately 3′ of        CH3 or M2;    -   5. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region excluding a CH1 gene segment;    -   6. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising a CH1 (and optionally also a CH2 and        CH3) gene segment;    -   7. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising a CH2 (and optionally also a CH3)        gene segment;    -   8. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising a CH3 gene segment;    -   9. A human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4)        constant region comprising CH2-CH3 gene segments and excluding a        CH1 gene segment;

Human Delta Constant Regions

-   -   10. A human delta constant region comprising or consisting of        the sequence from CH1 to (and including) CH3, CH-S, M1 or M2;    -   11. A human delta constant region comprising or consisting of        the sequence from hinge H1 or H2 to (and including) CH3, CH-S,        M1 or M2;    -   12. A human delta constant region comprising or consisting of        the sequence from CH2 to (and including) CH3, CH-S, M1 or M2;    -   13. A human delta constant region comprising or consisting of        the sequence from CH3 to (and including) CH-S, M1 or M2;    -   14. A human delta constant region excluding a CH1 gene segment;    -   15. A human delta constant region comprising a CH1 (and        optionally also a CH2 and CH3) gene segment;    -   16. A human delta constant region comprising a CH2 (and        optionally also a CH3) gene segment;    -   17. A human delta constant region comprising a CH3 gene segment;    -   18. A human delta constant region comprising CH2-CH3 gene        segments and excluding a CH1 gene segment;

Human Alpha Constant Regions

-   -   19. A human alpha constant region comprising or consisting of        the sequence from CH1 to (and including) CH3, M or the poly-A        immediately 3′ of M or CH3;    -   20. A human alpha constant region comprising or consisting of        the sequence from hinge (H) to (and including) CH3, M or the        poly-A immediately 3′ of M or CH3;    -   21. A human alpha constant region comprising or consisting of        the sequence from CH2 to (and including) CH3, M or the poly-A        immediately 3′ of M or CH3;    -   22. A human alpha constant region comprising or consisting of        the sequence from CH3 to (and including) M or the poly-A        immediately 3′ of M or CH3;    -   23. A human alpha constant region excluding a CH1 gene segment;    -   24. A human alpha constant region comprising a CH1 (and        optionally also a CH2 and CH3) gene segment;    -   25. A human alpha constant region comprising a CH2 (and        optionally also a CH3) gene segment;    -   26. A human alpha constant region comprising a CH3 gene segment;    -   27. A human alpha constant region comprising CH2-CH3 gene        segments and excluding a CH1 gene segment;

The alpha constant region is, in one example, a human alpha-1 constantregion. In another example the constant region is a human alpha-2constant region.

Human Epsilon Constant Regions

-   -   28. A human epsilon constant region comprising or consisting of        the sequence from CH1 to (and including) CH3, CH4, M or the        poly-A immediately 3′ of M or CH4;    -   29. A human epsilon constant region comprising or consisting of        the sequence from hinge (H) to (and including) CH3, CH4, M or        the poly-A immediately 3′ of M or CH4;    -   30. A human epsilon constant region comprising or consisting of        the sequence from CH2 to (and including) CH3, CH4, M or the        poly-A immediately 3′ of M or CH4;    -   31. A human epsilon constant region comprising or consisting of        the sequence from CH3 to (and including) CH4, M or the poly-A        immediately 3′ of M or CH4;    -   32. A human epsilon constant region comprising or consisting of        the sequence from CH4 to (and including) M or the poly-A        immediately 3′ of M or CH4;    -   33. A human epsilon constant region excluding a CH1 gene        segment;    -   34. A human epsilon constant region comprising a CH1 (and        optionally also a CH2, CH3 and CH4) gene segment;    -   35. A human epsilon constant region comprising a CH2 (and        optionally also a CH3 and CH4) gene segment;    -   36. A human epsilon constant region comprising a CH3 (and        optionally also a CH4) gene segment;    -   37. A human epsilon constant region comprising a CH4 gene        segment;    -   38. A human epsilon constant region comprising CH2-CH3-CH4 gene        segments and excluding a CH1 gene segment.

Multiple Isotype Constant Regions

-   -   39. A nucleotide sequence comprising or consisting of the        sequence from human gamma-3 to (and including) human gamma-4        constant regions;    -   40. A nucleotide sequence comprising or consisting of the        sequence from human gamma-3 to (and including) human gamma-2        constant regions;    -   41. A nucleotide sequence comprising or consisting of the        sequence from human gamma-3 to (and including) human gamma-1        constant regions;    -   42. A nucleotide sequence comprising or consisting of the        sequence from human gamma-1 to (and including) human gamma-4        constant regions;    -   43. A nucleotide sequence comprising or consisting of the        sequence from human gamma-1 to (and including) human gamma-2        constant regions;    -   44. A nucleotide sequence comprising or consisting of the        sequence from human gamma-2 to (and including) human gamma-4        constant regions;    -   45. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human alpha-2        constant regions;    -   46. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human epsilon        regions;    -   47. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human gamma-4        constant regions;    -   48. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human gamma-2        constant regions;    -   49. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human alpha-1        constant regions;    -   50. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human gamma-1        constant regions;    -   51. A nucleotide sequence comprising or consisting of the        sequence from human delta to (and including) human gamma-3        constant regions.

Stretches of human DNA can be inserted in one piece or in serialinsertions into the genome of a non-human vertebrate or cell (eg, EScell) genome. For example, a plurality of bacterial artificialchromosomes (BACs) can be constructed using standard recombineeringtechniques which between them comprise the entire sequence of humanconstant region DNA to be inserted, each BAC containing a portion ofthat stretch of DNA. By using serial insertions from serial BACs (eg,using standard homologous recombination (eg, see the Regeneron andAblexis PCTs disclosed herein) or serial recombinase mediated cassetteexchange—sRMCE—(eg, see the Kymab Limited PCTs disclosed herein) theskilled person can build up the one or more human constant regionsdownstream of the endogenous mu constant region in the genome. When anES cell is manipulated in this way, the ES cell can be used, as isstandard, to implant in a donor blastocyst, which is then implanted intoa foster mother. With germline transmission and any necessary subsequentbreeding and crossing (again as is conventional), the skilled person canarrive at progeny vertebrates and cells bearing the gene locus of theinvention in which one or a plurality of human non-mu constant regionsare downstream of an endogenous mu constant region. The gene locus maybe present in a homozygous state (eg, a gene locus of the invention ateach heavy chain allele) or heterozygous state (eg, with the secondallele being an inactivated endogenous heavy chain so that only heavychain expression from the first allele—bearing the gene locus of theinvention—provides expression of heavy chains). In an alternative,different gene loci of the invention are provided at the first andsecond alleles of the heavy chain locus in the genome, so that differentcombinations of human isotype switched heavy chains can be expressed bythe combination of loci.

In an embodiment, the genome also comprises transgenic light chain locithat express human light chains (or in the alternative at least humanlight chain variable regions) and endogenous light chain expression issubstantially inactive; also endogenous heavy chain expression issubstantially inactive. This embodiment advantageously provides a genomefor predictable expression of only fully human antibodies (or at leastfully human heavy chains and fully human light chain variable regions inthe alternative) and no endogenous antibody chains, from which pool ofantibodies the skilled person can select (eg, after immunisation withtarget antigen) non-mu type antibodies (eg, gamma type) that haveundergone mutation and maturation and selection by the in vivo machineryof the vertebrate or cell.

It is particularly beneficial to use embodiments in which one or morehuman gamma constant regions are inserted, since gamma-type antibodiesare frequently used in humans in immune responses in disease settingsand are thus frequently used as drugs for human medicine. Human gamma-1isotype is particularly useful for this reason, and many humantherapeutic antibody drugs have gamma-1 isotype constant domainsaccordingly.

As discussed further in WO2011066501, human IgG sub-types IgG1, IgG2,gG3 and IgG4 exhibit differential capacity to recruit immune functions,such as antibody-dependent cellular cytotoxicity (ADCC, e.g., IgG1 andIgG3), antibody-dependent cellular phagocytosis (ADCP, e.g., IgG1, IgG2,IgG3 and IgG4), and complement dependent cytotoxicity (CDC, e.g., IgG1,IgG3). Sub-type-specific engagement of such immune functions is based onselectivity for Fc receptors on distinct immune cells and the ability tobind C1q and activate the assembly of a membrane attack complex (MAC).Among the various types, relative affinity for Fcγ receptors (e.g.,FcγRI, FcγRIIa/b/c, FcγRIIIa/b) is high for IgG1 and IgG3, however,there is minimal affinity for IgG2 (restricted to the FcγRIIa 131Hpolymorphism), and IgG4 only has measurable affinity for FcγRI. Usingcomparative sequence analysis and co-crystal structures, the key contactresidues for receptor binding have been mapped to the amino acidresidues spanning the lower hinge and CH2 region. Using standard proteinengineering techniques, some success in enhancing or reducing theaffinity of an antibody preparation for Fc receptors and the C1qcomponent of complement has been achieved.

Among the isotypes, IgG2 is least capable of binding the family of Fcreceptors. Using IgG2 as the starting point, efforts have been made tofind a mutant with diminished effector functions but which retains FcRnbinding, prolonged stability, and low immunogenicity. Improved mutantsof this nature may provide improved antibody therapeutics with retainedsafety. Human IgG1 therapeutic antibodies that bind to cell surfacetargets are able to engage effector cells that may mediate cell lysis ofthe target cell by antibody-dependent cellular cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). These mechanisms occur throughinteraction of the CH2 region of the antibody Fc domain to FcγRreceptors on immune effector cells or with C1q, the first component ofthe complement cascade. Table 1 shows the activities of different humangamma sub-types. The skilled person may choose accordingly to promote ordampen-down activity depending upon the disease setting in humans ofinterest. For example, use of an insertion comprising human gamma-1constant region is desirable when one wishes to isolated totally humanheavy chains and antibodies that have relatively high complementactivation activity by the classical pathway and FcγR1 recognition inhuman patients.

TABLE 1 Summary of function correlated with human gamma sub-typeEffector functions of human IgG IgG1 IgG2 IgG3 IgG4 Complementactivation Classical pathway +++ + +++ − Alternative pathway − + − − Fcreceptor recognition FcγRI +++ − +++ ++ FcγRIIa, 131R/R ++ − ++ −FcγRIIa, 131H/H + + ++ − FcγRIIb ++ − ++ + FcγRIII + +/− + +/−

See also Mol Immunol. 2003 December; 40(9):585-93; “Differential bindingto human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype andmutant antibodies”; Armour K L et al, which is incorporated herein byreference.

IgG2 constant regions are well suited to producing antibodies and heavychains according to the invention for binding to cytokines or solubletargets in humans, since IgG2 is essentially FcγRI,III-silent,FcγRIIa-active and has little Complement activity.

IgG1 constant regions have wide utility for human therapeutics, sinceIgG1 antibodies and heavy chains are FcγRI,II,III-active and havecomplement activity. This can be enhanced by using a human gamma-1constant region that has been activated by engineering as is known inthe art.

Thus, in one embodiment of the vertebrate or cell of the invention thehuman non-mu constant region comprises CH2 or CH2 and CH3 gene segmentsencode for an activated Fc region. This is useful for, eg, treating orpreventing inflammation or a inflammatory disease or condition. Thisembodiment is also useful where cell killing in the human patient isdesirable, eg, for oncology applications.

In one embodiment of the vertebrate or cell of the invention the humannon-mu constant region comprises CH2, or CH2 and CH3, gene segmentsencode for an inactivated Fc region. This is useful for, eg,applications where triggering following cross-linking is undesirable.This may be useful, for example where cross-linking of cell-surfaceantigens is to be avoided after administration of an antibody producedaccording to the invention (eg, where the target antigen against whichthe antibody or heavy chain is raised is a T-cell costimulatory antigensuch as CD40 or CD40 ligand).

The choice of constant region and gene segments to include in the humanconstant region(s) may also take into account the desirability for goodbinding to human FcRn in the human patient, so that the heavy chain orantibody comprising this is efficiently recycled and a desirable drughalf-life is achieved. For this purpose, gamma-type human constantregions are desirable, since IgG antibodies and heavy chains bindefficiently to human FcRn in vivo.

“Antibody-dependent cell-mediated cytotoxicity” or ADCC″ refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) that enables these cytotoxic effectorcells to bind specifically to an antigen-bearing target cell andsubsequently kill the target cell with cytotoxins. Ligand specifichigh-affinity IgG antibodies directed to the surface of target cellsstimulate the cytotoxic cells and are required for such killing. Lysisof the target cell is extracellular, requires direct cell-to-cellcontact, and does not involve complement.

The ability of any particular antibody to mediate lysis of the targetcell by ADCC can be assayed. To assess ADCC activity, an antibody ofinterest is added to target cells displaying the target ligand incombination with immune effector cells, which may be activated by theantigen antibody complexes resulting in cytolysis of the target cell.Cytolysis is generally detected by the release of label (e.g.,radioactive substrates, fluorescent dyes or natural intracellularproteins) from the lysed cells. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Specific examples of in vitro ADCC assays are described inWisecarver et al, 1985, 19:21 1; Bruggemann et al, 1987, J Exp Med 166:1351; Wilkinson et al, 2001, J Immunol Methods 258: 183; Patel et al,1995 J Immunol Methods 184:29 (each of which is incorporated byreference). Alternatively, or additionally, ADCC activity of theantibody of interest may be assessed in vivo, e.g., in an animal model,such as that disclosed in Clynes et al, 1998, PNAS USA 95:652, thecontents of which are incorporated by reference in its entirety.

“Complement-directed cytotoxicity” or CDC refers to the form ofcytotoxicity in which the complement cascade is activated by thecomplement component C1q binding to antibody Fc.

The term “Fc” as used herein refer to the protein comprising (in N- toC-terminal direction) an immunoglobulin CH2 and CH3 domain. The CH2 andCH3 domains can form at least a part of the constant region of anantibody or heavy chain.

“Constant region” as used herein when referring to the gene locus of theinvention, is a reference to a stretch of DNA sequence comprising genesegments (constant region gene segments) that combine in vivo withrecombined VDJ sequence in the locus. The VDJ yield (optionallyfollowing hypermutation), together with the constant region genesegments RNA transcripts in the vertebrate or cell from which can beproduced antibody heavy chains (eg, following splicing of RNA), eachheavy chains comprising an variable domain and one or more constantdomains. When “constant region” is used with reference to an antibody orantibody heavy chain, this is referring to the region C-terminal to thevariable domain and comprising one or more constant domains.

“Endogenous” as used herein indicates that the constant region etc is atype of constant region etc that is normally found in the vertebrate orcell (as opposed to an exogenous constant region whose sequence is notnormally found in such a vertebrate or cell, eg a human sequence). Forexample, the endogenous constant region can be those encoded by thewild-type genome of the non-human vertebrate or cell. So, in an examplewherein the vertebrate is a mouse, the endogenous constant region wouldbe a mouse constant region. Going further, the endogenous regions are,in an example, strain-matched to the vertebrate or cell. So, in oneembodiment, the vertebrate or cell is a mouse 129 ES cell, theendogenous constant region would be mouse 129 constant region. Inanother embodiment, the vertebrate or cell is a JM8 strain mouse ormouse cell, the endogenous constant region would be mouse JM8 constantregion. In another embodiment, the vertebrate or cell is a Black 6 mouseor mouse cell, the endogenous constant region would be mouse Black 6constant region.

In an embodiment, the gene locus in the vertebrate or cell of theinvention comprises (in 5′ to 3′ direction) one or moremembrane-encoding exons (M) and a polyA downstream of the non-humanhuman constant region gene segments, wherein the vertebrate is capableof expressing membrane-bound heavy chain or antibody of said non-muisotype. Additionally or alternatively, the locus comprises a polyAimmediately downstream of the last human constant region gene segment.The locus may further comprise a splice site between the latter polyAand the one or more membrane-encoding exons, wherein the vertebrate orcell is capable of expressing both membrane-bound and secreted forms ofthe non-mu heavy chain or antibody.

In order to obtain switching from the endogenous mu to human non-muisotypes, the locus comprises a switch 5′ of the mu constant region (eg,the endogenous S-mu is retained) and a second switch 5′ of the (or each)human non-mu constant region. The second switch can be the human switchusually present with the human constant region (eg, when the humanconstant region is a gamma-1 constant region, the human gamma-1 switchis retained). Alternatively, the switch can be the correspondingendogenous non-human vertebrate switch, in this example an endogenousS-gamma (eg, S-gamma-1), eg, where a nucleotide sequence correspondingto human DNA from CH1 to the M2 or polyA 3′ of M2 of a human gammaconstant region is inserted downstream of an endogenous S-gamma in thevertebrate or cell genome. Switch sequences are known in the art, forexample, see Nikaido et al, Nature 292: 845-848 (1981) and alsoWO2011004192, U.S. Pat. Nos. 7,501,552, 6,673,986, 6,130,364,WO2009/076464 and U.S. Pat. No. 6,586,251, eg, SEQ ID NOs: 9-24disclosed in U.S. Pat. No. 7,501,552. Commercial human and mouse BAClibraries (eg, the RPC-11 and Caltech A, B, C and D libraries) aresources of suitable immunoglobulin locus sequences, eg, sequences ofswitches or constant regions. Alternatively, human DNA samples can beobtained de novo from consenting donors (eg, using cheek swabs) and DNAsequences obtained from the samples.

In an embodiment, the vertebrate or cell of the invention comprises anantibody heavy chain locus that comprises (in 5′ to 3′ direction) arearranged or unrearranged variable region, a first switch (eg, an S-muswitch, eg an endogenous S-mu), an endogenous mu constant region, asecond switch (an S-gamma switch; eg, endogenous S-gamma) and a humanconstant region of said non-mu isotype; wherein the heavy chain locus iscapable of undergoing switching from IgM to the non-mu isotype. Thus,for example, this can be provided by inserting one or more human non-muconstant region sequences downstream (3′) of an endogenous mu region inthe genome of a non-human vertebrate or cell (eg, ES cell). In this way,the human sequences are provided within the location of the endogenousheavy chain locus and expression from the human sequence can beeffectively controlled using endogenous antibody and B-cell mechanisms.

By accommodating VH, D and JH recombination and initial expression inthe context of an endogenous IgM, heavy chain variable regions areproduced and selected in context of non-human (eg, mouse) mu constantregions. This advantageously enables endogenous RAG-1 and RAG-2 to beefficiently employed in a completely endogenous context, as well asharnessing endogenous control, mutation and signalling involved inantibody and B-cell development. The invention subsequently enablesswitching to a human non-mu C region, which enables one to produce humanconstant domains in product heavy chains which have been affinitymatured by somatic hypermutation effected by endogenous activationinduced cytidine deaminase (AID). Those rearranged variable regions thatstill work well (eg, good expression, affinity, biophysicalcharacteristics) in the context of the human C domains are selected invivo by the mouse and amplified following immunisation with apredetermined antigen. This retains the advantage of harnessingendogenous antibody and B-cell production and control, together withaffinity maturation and in vivo amplification of selected B-cellsexpressing desirable antibodies/heavy chains in a way that harnesses themouse (or other non-vertebrate) system to select for good, fully-humanantibodies and heavy chains. This is superior to the prior art method ofhumanising in vitro, where selection for good biophysicalcharacteristics, expression, affinity etc is not factored in (thus oftenresulting in a down-grading of these in the prior art) and which addsmulti-step procedures following initial production of a chimaericantibody in vivo.

In one embodiment, the vertebrate or cell of the invention does notexpress endogenous antibody heavy chains of said non-mu isotype. Forexample, this is effected using standard methods to delete or inactivateendogenous heavy chain variable and constant regions. Thus, the skilledperson will know that heavy chains (and antibodies comprising these) ofthe predetermined human non-mu isotype (eg, IgG antibodies) produced bythe vertebrate or cell will always comprise human constant regions.

In one embodiment, in the vertebrate or cell of the invention the humannon-mu constant region is a gamma constant region (eg, gamma-1, -2, 3 or-4). Instead of a gamma constant region, the constant region can be adelta, alpha (eg, alpha-1 or -2) or epsilon constant region. Thus, theinvention allows the skilled person to tailor the genome of thevertebrate or cell according to desired constant region functionality ofresultant antibodies and heavy chains, as discussed above.

In one embodiment, in the vertebrate or cell of the invention the gammaconstant region comprises CH2 and CH3 gene segments for encoding anantibody Fc region. In an example, the constant region also comprises aCH1 gene segment of the human constant region. This is useful forproducing heavy chains that are to form part of 4-chain antibodies(classic H2L2 antibodies). In another example, the constant region lacksa CH1 gene segment. This is useful for producing heavy chains that areto form part of heavy-chain only antibodies (H2 antibodies) which aredevoid of light chains.

In one embodiment, in the vertebrate or cell of the invention the genomecomprises a light chain locus comprising human VL and JL gene segmentsupstream of a constant region for expression of light chains comprisinghuman variable regions; optionally wherein the constant region is ahuman light chain constant region. For example, the genome comprises alight chain locus comprising human Vκ and Jκ gene segments upstream ofan endogenous or human kappa constant region in the endogenous kappalocus for expression of kappa light chains comprising human variableregions. In this example, where the vertebrate is a mouse, it may not benecessary to humanise the lambda locus, since lambda chain expression inmice is relatively low. Optionally, the lambda locus is inactive and/orcomprises human Vλ and Jλ upstream of an endogenous or human lambdaconstant region. Optionally, endogenous kappa chain expression isinactive.

For example, the vertebrate or cell does not substantially expressendogenous light chains.

Inactivation of endogenous antibody, antibody chain or gene segmentusage is, for example, substantially complete inactivation or prevention(substantially 100%, ie, essentially none (eg, less than 10, 5, 4, 3, 2,1 or 0.5%) of the endogenous antibody chain etc (eg, no endogenous heavychains etc) is expressed). This can be determined, for example, at theantibody chain (protein) level by assessing the antibody repertoireproduced by the non-human vertebrate or cell or at the nucleotide levelby assessing mRNA transcripts of antibody gene loci, eg, using RACE. Inan embodiment, inactivation is more than 50% (ie, 50% or less of theantibodies or transcripts are of an endogenous antibody chain), 60%,70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. For example, in anembodiment, endogenous heavy chain expression is substantiallyinactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99%of the heavy chain repertoire of the vertebrate is provided byendogenous heavy chains. For example, endogenous heavy chain expressionis substantially inactivated such that substantially none of the heavychain repertoire of the vertebrate is provided by endogenous heavychains. For example, in an embodiment, endogenous kappa chain expressionis substantially inactivated such that no more than 85%, 90%, 95%, 96%,97%, 98% or 99% of the kappa chain repertoire of the vertebrate isprovided by endogenous kappa chains. For example, endogenous kappa chainexpression is substantially inactivated such that substantially none ofthe kappa chain repertoire of the vertebrate is provided by endogenouskappa chains. For example, in an embodiment, endogenous lambda chainexpression is substantially inactivated such that no more than 85%, 90%,95%, 96%, 97%, 98% or 99% of the lambda chain repertoire of thevertebrate is provided by endogenous lambda chains. For example,endogenous lambda chain expression is substantially inactivated suchthat substantially none of the lambda chain repertoire of the vertebrateis provided by endogenous lambda chains.

In one embodiment, in the vertebrate or cell of the invention the humangamma constant region does not comprise a CH1 gene segment. In thisembodiment, in an example, light chain expression is inactive when thenon-mu heavy chains are expressed. This is useful for producingheavy-chain only antibodies of the non-mu isotype.

In one embodiment, in the vertebrate or cell of the invention the locuscomprises a replacement of endogenous non-mu constant region genesegments (eg, CH2 and CH3 gene segments) with corresponding humanconstant region gene segments (eg, CH2 and CH3 gene segments) of thesame non-mu isotype; optionally wherein an endogenous switch of saidnon-mu isotype is retained. This may be advantageous for maintaining thenon-mu switch control of switching to the accompanying human isotype.Replacement here means functional or physical replacement, wherein theendogenous constant region of said non-mu isotype is non-functional orabsent in the final genome and the human constant region is functionaland present.

The vertebrate or cell of any preceding claim, wherein the locuscomprises an insertion of a human gamma constant region nucleotidesequence comprising sequence from the human gamma S-gamma CH1, hinge orCH2 gene segment to (and including) the human gamma CH3 gene segment,wherein the insertion replaces a corresponding sequence of an endogenousgamma constant region in the genome of the vertebrate or cell. This isadvantageous for maintaining the human CH2 and CH3 and inter-genesegment sequences in relative genomic or germline configuration forproper expression of human non-mu Fc in the product heavy chain orantibodies. Proper Fc function is desirable for Fc receptor interactionsand recycling in human patients receiving the chain or antibody or aderivative drug thereof, as discussed above. Replacement here meansfunctional or physical replacement, wherein the endogenous constantregion of said non-mu isotype is non-functional or absent in the finalgenome and the human constant region is functional and present.

In an example, the human constant region is in human germlineconfiguration. The term “germline configuration” refers to a germlinegenomic configuration. For example, human immunoglobulin gene segmentspresent in the gene locus of the invention are in a germlineconfiguration when the relative order of the human gene segments is thesame as the order of corresponding gene segments in a human germlinegenome. For example, when the gene locus of the invention compriseshypothetical human immunoglobulin constant region gene segments A, B andC, these would be provided in this order (5′ to 3′ in the locus) whenthe corresponding gene segments of a human germline genome comprises thearrangement 5′-A-B-C-3′; and optionally the inter-gene segments too arein germline configuration (ie, the nucleotide sequence from A to Ccorresponds to a stretch of contiguous nucleotide sequence from A to Cin a human germline genome). Available databases and sources of humangermline sequence are discussed below. Thus, in an example, whenelements of a human immunoglobulin locus (eg, gene segments, enhancersor other regulatory elements) are provided in a gene locus according tothe invention, the human Ig locus elements are in germline configurationwhen the relative order of the elements is the same as the order ofcorresponding elements in a human germline genome and human sequencesbetween the elements are included, these corresponding to such sequencesbetween corresponding elements in the human germline genome. Thus, in ahypothetical example the transgenic locus comprises human elements inthe arrangement 5′-A-S1-B-S2-C-S3-3′, wherein A, B and C are humanimmunoglobulin gene segments and S1-S3 are human inter-gene segmentsequences, wherein the corresponding arrangement 5′-A-S1-B-S2-C-S3-3′ ispresent in a human germline genome. For example, this can be achieved byproviding in a transgenic immunoglobulin locus of the invention a DNAinsert corresponding to the DNA sequence from A to C in a human germlinegenome (or the insert comprising the DNA sequence from A to C). Thearrangements in human germline genomes and immunoglobulin loci are knownin the art (eg, see the IMGT, Kabat and other antibody resources).

In an embodiment of the invention, endogenous inter-gene segmentsequences have been retained so that the heavy chain locus comprisessaid human non-mu constant region gene segments flanked by endogenousconstant region inter-gene segment sequences. This is useful forpreserving any endogenous, non-human regulatory elements between genesegments—for control using endogenous mechanisms for class switching andantibody/chain production in the vertebrate or cell.

In an embodiment of the invention, the gene locus comprises areplacement of endogenous gamma constant region gene segments (eg, gammaCH2 and CH3 gene segments) with human gamma constant region genesegments (eg, gamma CH2 and CH3 gene segments), but endogenous sequencesbetween gamma constant regions have been retained. This is useful forpreserving any endogenous, non-human regulatory elements between genesegments—for control using endogenous mechanisms for class switching andantibody/chain production in the vertebrate or cell.

Populations for Providing Alternative Combinations & In Vivo Selectionsof Human Constant Regions

The invention also provides a general combinatorial or repertoireapproach that enables the skilled person to design an antibody/heavychain production and selection scheme that combines human mutatedvariable regions with a plurality of different human constant domains(and/or synthetic mutants thereof). This scheme straight-forwardlyharnesses the in vivo non-human vertebrate system to performcombinations, affinity maturation, and selection by expression andamplification of target antigen-specific heavy chains that areproductive and in a fully human configuration. Thus, the skilled personis provided with a way of obtaining a repertoire of matured, targetantigen-specific human heavy chain variable domains paired with arepertoire of human constant regions. The skilled person will know thatall members of the repertoire represent productive combinations of humanvariable and constant heavy chain regions, in that they can be expressedto detectable and selectable levels in an in vivo system. From therepertoire (eg, a repertoire provided in a population of non-humanvertebrates of the invention; or a repertoire of B-cells isolated fromsuch vertebrates; or a repertoire of antibodies or heavy chains isolatedfrom such vertebrates or cells; or a repertoire of nucleotide sequencesencoding such antibodies or heavy chains) the skilled person is able toselect one or more antibodies or heavy chains (or correspondingnucleotides sequences) that bind the target antigen. Selection isperformed on the basis of a desired antibody/chain characteristic, suchas antigen binding affinity and/or a biophysical characteristic.

To this end, there is provided a population of vertebrates according tothe invention, wherein the population comprises

(i) a first of said vertebrates wherein said gene locus is an antibodyheavy chain locus that comprises one or more first human non-mu genesegment(s) (eg, a human CH1, CH2 or CH3; or an Fc) or a first humannon-mu constant region;(ii) and a second of said vertebrates wherein said gene locus is anantibody heavy chain locus that comprises one or more second humannon-mu gene segment(s) (eg, a human CH1, CH2 or CH3; or encoding an Fc)or a second human non-mu constant region; andwherein the first and second gene segments are the same type of constantregion gene segment(s) (eg, both CH1 gene segments or both encoding anFc) and the second gene segment(s) is a variant of (ie, is a mutant of)the first gene segment(s).

Thus, this aspect of the invention is useful, for example, because itenables the skilled person to use the vertebrates to select fordesirable human V regions (and those that express and perform well, havegood biophysical characteristics etc) in vivo in the context of arepertoire of naturally-occurring human constant region polypmorphicvariants—thus one can include the most common human variants (eg, aspresent in the 1000 Genomes database; www.1000genomes.org) to arrive athuman antibodies and heavy chains that have wide application in humanmedicine, or can be selected (according to the C region variantdistribution in the human ethnic and other populations) to be tailoredto specific human populations. Also variants may have differingexpression and/or biophysical characteristics—thus allowing one to havea pool of human antibodies and heavy chains from which to select gooddrug candidates for human use.

Thus, in an example, the second gene segment(s) is derived from thegenome sequence of different human individuals, for example sequencesappearing in the 1000 Genomes database (www.1000genomes.org).

The variant can, in one example, have no more than 15, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 mutations compared to the corresponding first genesegment(s) or region.

In an embodiment, the second gene segment(s) or region is a syntheticmutant of the first gene segment(s) or region. In one example, thesecond gene segment(s) or region has no more than 15, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 mutations compared to the corresponding first genesegment(s) or region. In this way, the skilled person can test outmutations in vivo in combination with a repertoire of human heavy chainvariable regions. Combinations that are not productive will not beexpressed and/or proliferate (as regulated by the vertebrates' own invivo machinery) and productive combinations are then provided in one ofsaid repertoires for selection of one or more desirable antibodies orheavy chains bearing human constant regions. Thus, for example, one canmake (eg, design by specific choice of positions to mutate and/orresidues to be encoded) mutant variants of a desired type of constantregion (eg, to produce a collection of constant region encoding variantgamma, eg, gamma-1, constant regions). The mutants, in one example, aremade with the aim of finding Fc inactivating variants that work wellwith the selected human variable regions. In another example, themutants are made with the aim of finding Fc activating variants thatwork well with the selected human variable regions. Inactivation oractivation of Fc can be tested (as is known in the art) after one ormore antibodies or heavy chains have been selected from the populationor repertoire. Advantageously, no further manipulation is required invitro and expressibility of mutants and combinations is guaranteed in anin vivo system (at least cells of the non-human vertebrate type). Forexample, by immortalising isolated B-cells expressing to the selectedantibodies/heavy chains (eg, by immortalising the B-cell directly or byfusion with a myeloma cell to produce a hybridoma), one then has a cellline that provides a good source of selected antibodies. Additionally,since in vitro manipulation is not necessary post-selection, the risk ofdown-grading biophysical characteristics is lowered and one can simplytest the biophysical characteristics of a panel of lead antibodies/heavychains (that have been selected for antigen binding in the desiredaffinity range) and select one or more antibodies/chains from this panethat meet the profile of characteristics required for human medicinaluse and drug production and administration methods.

Thus, in an embodiment, one or both of the first and second genesegment(s) or regions encodes an inactivated human gamma Fc.

In an embodiment one or both of the first and second gene segment(s) orregions encodes an activated human gamma Fc.

Thus, the population of the invention provides an overall heavy chainrepertoire comprising the heavy chain sequence repertoires of the firstand second vertebrates. The vertebrates in the population can beimmunised with the same antigen in a method of selecting and isolatingone or more heavy chains (eg, provided as part of antibodies) thatspecifically bind to the antigen.

The VH gene segments of a repertoire can, in one embodiment, berecombined VH, ie, provided as part of a variable region sequencederived from the recombination of human VH with D and JH (eg, where theVH, D and JH are human).

In an embodiment, the population comprises a third said non-humanvertebrate, wherein the human constant region gene segment repertoire isdifferent from those of the first and second vertebrates, whereby thethird vertebrate can produce a heavy chain sequence repertoire that isdifferent from the heavy chain sequence repertoire produced by the firstand second vertebrates. Thus, the population provides an overall heavychain repertoire comprising the heavy chain sequence repertoires of thefirst, second and third vertebrates. The vertebrates in the populationcan be immunised with the same antigen in a method of selecting andisolating one or more heavy chains (eg, provided as part of antibodies)that specifically bind to the antigen.

In an embodiment, VH gene segment repertoire provided by said populationcomprises a substantially complete repertoire of functional human VHgene segments; optionally providing at least 6 different human JH genesegments, 27 different human D segments and at least 40 different humanVH gene segments.

In an embodiment, the VH gene segment repertoire provided by saidpopulation comprises at least 20, 25, 30, 35 or 40 different human VHgene segments.

In an embodiment, the method of the invention comprises the step ofimmunising the vertebrates of the population with the same antigen (eg,a human antigen). Thus, the vertebrates are a population and are used assuch.

In an embodiment, immunisation of two, more or all of said vertebratesis separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or1 week or 6, 5, 4, 3, 2, or 1 day. Thus, the vertebrates are apopulation and are used as such.

In an embodiment, the method of the invention comprises the step ofselecting one or more heavy chains or antibodies from each of saidimmunised vertebrates on the basis of a common desired antibody or heavychain characteristic (eg, binding affinity for said antigen), whereinthe selected antibodies comprise human non-mu heavy chains, or theselected heavy chains are human.

In an example, the vertebrates share the same genetic background, withthe exception of the heavy chain loci thereof (and optionally one ormore of the light chain loci thereof).

In any configuration of the invention, in an embodiment vertebrates arederived from transgenic non-human vertebrate ancestor embryonic stemcells that have been genetically modified to include humanimmunoglobulin locus DNA, the ancestor stem cells being identical orrelated (eg, clonally related); optionally wherein the genome of theancestor stem cells comprise a common sequence junction that is ajunction between a non-human vertebrate sequence and a human sequence(eg, the ancestor genomes comprise a common transgenic immunoglobulinlocus or a common human/non-human vertebrate (eg, human/mouse orhuman/rat) DNA junction). For example, the genomes comprise a commonjunction within or at the boundary of one or more of theirimmunoglobulin chain loci (eg, heavy chain loci and/or light chainloci). For example, the vertebrates of the population are mice whosegenomes comprise a common human-mouse DNA junction within their heavychain loci and/or one or more light chain loci. This is indicative thatthe mice form a population. For example, by producing variantvertebrates all stemming back from a common ancestor, the vertebratescan all share the same genetic background with the exception of one ormore human gene segment repertoires in their genomes. This means that,with the exception of the expression profile resulting from thedifferent gene segment sub-repertoires, there are no other introducedgenetic variables between the members of the population, which enhancesconsistency of performance of the members of the population. This alsosimplifies breeding to produce the variants making up the population.

The invention provides an animal house or a laboratory containing apopulation according to the invention. For example, vertebrates of thepopulation can be housed in the same cage or in the same collection ofcages in the same animal house, building or laboratory. The cages orvertebrates themselves may be labelled so that they are part of the samepopulation or experiment. They may be owned by the same owner, eg, thesame company, or in the control of a single person or company. They maybe allocated for use in the same research programme or series of relatedresearch experiments aimed at discovering one or more antibodies orantibody chains against a common antigen or related antigens. Thus, thevertebrates provide a population and are used as such. It is indicativeof a population, that the vertebrates are discussed in the context ofthe same research programme or immunisation schedule or experiment orset of experiments in a laboratory notebook or a set of laboratorynotebooks that relate to the same research programme or immunisationschedule or experiment or set of experiments. For example, such aprogramme, schedule or experiment(s) may relate to immunisation of thevertebrates of a population with the same antigen.

Methods of In Vivo Humanisation & Antibody/Chain Selection

An aspect provides a method of isolating an antibody, heavy chain ornucleotide sequence encoding said antibody, the method comprising

(a) immunising (see e.g. Harlow, E. & Lane, D. 1998, 5^(th) edition,Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press,Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the NationalAcademy of Sciences (2004) 101:257-259) a vertebrate or populationaccording to the invention with an antigen such that the vertebrate(s)produce antibodies; and(b) isolating from immunised vertebrate(s) an antibody or heavy chainthat specifically binds to said antigen and/or a nucleotide sequenceencoding at least the heavy chain variable region of said antibody.

Suitably an immunogenic amount of the antigen is delivered in the methodof the invention. The invention also relates to a method for detecting atarget antigen comprising detecting an antibody or heavy chain producedas above or a derivative thereof with a secondary detection agent whichrecognises a portion of that antibody/chain.

Isolation of the antibody in step (b) can be carried out usingconventional antibody selection techniques, eg, panning for antibodiesagainst antigen that has been immobilised on a solid support, optionallywith iterative rounds at increasing stringency, as will be readilyapparent to the skilled person.

As a further optional step, after step (b) the amino acid sequence ofthe heavy chain variable region is mutated to improve affinity forbinding to said antigen. Mutation can be generated by conventionaltechniques as will be readily apparent to the skilled person, eg, byerror-prone PCR. Affinity can be determined by conventional techniquesas will be readily apparent to the skilled person, eg, by surfaceplasmon resonance, eg, using Biacore™.

Additionally or alternatively, as a further optional step, after step(b) the amino acid sequence of the heavy chain variable region ismutated to further improve one or more biophysical characteristics ofthe antibody, eg, one or more of melting temperature, solution state(monomer or dimer), stability and expression (eg, in CHO or E. coli).

An aspect provides an antibody or heavy chain produced by the method ofthe invention, optionally for use in human medicine, eg, for treatingand/or preventing a medical condition or disease in a human patient.

The invention provides a method of isolating a human antibody, themethod comprising immunising a vertebrate or population of vertebratesaccording to any preceding claim with an antigen (eg, human antigen orviral or bacterial antigen) and isolating an antibody from saidvertebrate or a vertebrate of the population, wherein the isolatedantibody specifically binds to the antigen and comprises a fully humanheavy chain of said non-mu isotype. The invention provides apharmaceutical composition comprising the isolated antibody or a copy orderivative thereof.

The method provides a method of obtaining a humanised and affinitymatured antigen-specific antibody heavy chain, the method comprisinghumanising the heavy chain in vivo in a non-human vertebrate (eg, amouse or a rat) comprising a functional activation induced cytidinedeaminase (AID) by immunising the vertebrate with the antigen andobtaining somatic hypermutation and isotype switching in a B-cell of thevertebrate from an endogenous mu isotype to a human non-mu isotype,wherein an affinity matured antigen-specific antibody heavy chain isproduced and expressed by the vertebrate, the non-mu constant domains ofthe heavy chain being human constant domains, the method furthercomprising isolating said humanised heavy chain.

In an example, the variable domain of the heavy chain is a humanvariable domain.

In an example, the vertebrate is according to any aspect of theinvention as described above.

The method provides a pharmaceutical composition comprising the isolatedantibody heavy chain obtained in the method or a copy or derivativethereof; optionally wherein the heavy chain, copy or derivative isprovided by an antibody that specifically binds the antigen.

Optionally, the method comprises the step of isolating a B-cell fromsaid immunised vertebrate (or an immunised vertebrate of saidpopulation), wherein the B-cell expresses said isolated antibody orheavy chain; and optionally immortalising the B-cell.

Optionally, the method comprises the step of isolating a nucleotidesequence from said immunised vertebrate or B-cell, wherein thenucleotide sequence encodes said isolated antibody or a heavy chainthereof.

The method provides a vector (optionally in a host cell) comprising thenucleotide sequence of the invention or a copy thereof, or a derivativethereof; optionally comprising up to 15, 10, 9, 8, 7, 6, 5, 4, 3, or 1mutations. The derivative specifically binds target antigen.

The method provides a method of treating or preventing a medicalcondition or disease in a human associated or caused by said antigen,the method comprising administering to the human the antigen-specificantibody or heavy chain obtained in the method.

The method provides the antigen-specific antibody or heavy chainobtained in the method for use in the treatment or prophylaxis of amedical condition or disease in a human associated or caused by saidantigen.

The method provides the use of the antigen-specific antibody or heavychain obtained in the method in the manufacture of a medicament for usein the treatment or prophylaxis of a medical condition or disease in ahuman associated or caused by said antigen.

As will be readily apparent to the skilled person, the operableconnection of a human gene segment (eg, a V or J gene segment) upstreamof a constant region in an Ig locus in any configuration of theinvention enables the gene segment to be recombined and expressed in animmunoglobulin chain comprising sequence encoded by the constant regionof the locus.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, as well as antibody fragments (e.g., Fab,F(ab′)2). The term “antibody” also includes H2 antibodies that comprisea dimer of a heavy chain (5′-VH-(optional Hinge)-CH2-CH3-3′) and aredevoid of a light chain (akin to naturally-occurring H2 antibodies; see,eg, Nature. 1993 Jun. 3; 363(6428):446-8; Naturally occurring antibodiesdevoid of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S,Robinson G, Hamers C, Songa E B, Bendahman N, Hamers R). Thus, in anembodiment of the present invention, RNA produced from the transgenicheavy chain locus of the invention encodes for heavy chains that redevoid of a CH1 gene segment and comprise no functional antibody lightchain. In an example, RNA produced from the transgenic heavy chain locusencodes for VH single variable domains (dAbs; domain antibodies) withone or more human constant domains. Antibodies, according to theinvention, comprise one or more human constant domains of non-muisotype.

Examples of antibodies are classic 4-chain antibodies comprising twoheavy chains paired with two light chains (such as, a dimer of5′-VH-CH1-Hinge-CH2-CH3-3′ paired with 5′-VL-CL-3′) or H2 antibodiesthat comprise a dimer of a heavy chain (5′-VH-(optionalHinge)-CH2-CH3-3′) and are devoid of a light chain Thus, in anembodiment of the present invention, the heavy chain sequence repertoireencodes for heavy chains that re devoid of a CH1 gene segment andcomprise no functional antibody light chain. In an example, the heavychain sequence repertoire encodes a repertoire of VH single variabledomains (dAbs; domain antibodies) with one or more human constantdomains.

In an example of any configuration of the invention, a repertoirecomprises a plurality of different members (thus, for example, a heavychain repertoire comprises a plurality of different heavy chainsequences, such as sequences differing in their variable regions and/orhuman non-mu constant regions). In an example of any configuration ofthe invention, a repertoire comprises or consists of 2, 3, 4, 5, 6, 7,8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, atleast 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹, at least 10¹², atleast 10¹³, or at least 10¹⁴ members. For example, a repertoire ofantibody heavy chains or antibodies comprises or consists of at least100, at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹, at least10¹², at least 10¹³, or at least 10¹⁴ antibody chain sequences orantibodies respectively. For example, a repertoire comprises or consistsof 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 10³, at least 10⁴, at least 10⁵, at least10⁶, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least10¹¹, at least 10¹², at least 10¹³, or at least 10¹⁴ different members.For example, a repertoire of gene segments comprises or consists of 2,3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least40, at least 50 gene segments. Optionally, all of the gene segments aredifferent from each other; or all of the gene segments of the same type(eg, CH2) are different from each other.

In an example of any configuration of the invention, a populationcomprises a plurality of different members. Thus, for example, apopulation of non-human vertebrates (eg, mice or rats) comprises aplurality of vertebrates wherein at least two or more of the vertebratescomprise non-identical genomes. The genomes differ in their respectiverepertoire of human constant region gene segments. In an example of anyconfiguration of the invention, a population of non-human vertebratescomprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 10³ non-humanvertebrates. When the population consists of two vertebrates, thegenomes of vertebrates are different.

In one embodiment in any configuration of the invention, each vertebrateis a non-human mammal. In one embodiment in any configuration of theinvention, the vertebrate is a mouse, rat, rabbit, Camelid (eg, a llama,alpaca or camel), chicken, lamprey or shark. For example, allvertebrates are of the same vertebrate species, eg, all mice or allrats.

In any configuration of the invention, the human gene regions orsegments may be derived from the same individual or differentindividuals, or be synthetic (synthetically mutated human gene segments)or represent human consensus sequences.

Techniques for constructing non-human vertebrates and vertebrate cellswhose genomes comprise a human immunoglobulin gene segments are wellknown in the art. For example, reference is made to WO2011004192, U.S.Pat. Nos. 7,501,552, 6,673,986, 6,130,364, WO2009076464 and U.S. Pat.No. 6,586,251, the disclosures of which are incorporated herein byreference in their entirety.

In one example, a non-human vertebrate of any configuration of theinvention is able to generate a diversity of at least 1×10⁶ differentfunctional non-mu immunoglobulin sequence combinations.

Human variable regions are suitably inserted upstream of a non-humanvertebrate mu constant region, the latter together with downstream humanconstant region(s) comprising all of the DNA required to encode the fullconstant region or a sufficient portion of the constant region to allowthe formation of an effective non-mu antibody/heavy chain capable ofspecifically recognising a target antigen.

The endogenous mu non-human vertebrate constant region herein isoptionally the endogenous host wild-type constant region located at thewild type locus, as appropriate for the heavy chain. For example, thehuman constant region DNA is suitably inserted on mouse chromosome 12,immediately downstream of the mouse heavy chain mu constant region,where the vertebrate is a mouse.

In one optional aspect where the vertebrate is a mouse, the insertion ofhuman variable region DNA (V, D and J gene segments) is targeted to theregion between the J4 exon and the endogenous Sμ locus in the mousegenome IgH locus, and in one aspect is inserted between coordinates114,667,090 and 114,665,190, suitably at coordinate 114,667,091, after114,667,090.

All nucleotide co-ordinates for the mouse are those corresponding toNCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEMBL Release55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 seewww.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQ.releases.html)unless otherwise specified. Human nucleotides coordinates are thosecorresponding to GRCh37 (e.g. UCSC version hg 19,http://genome.ucsc.edu/FAQ/FAQ.releases.html), February 2009 ENSEMBLRelease 55.or are those corresponding to NCBI36, Ensemble release 54unless otherwise specified. Rat nucleotides are those corresponding toRGSC 3.4 Dec. 2004 ENSEMBL release 55.34w, or Baylor College of MedicineHGSC v3.4 Nov. 2004(e.g., UCSC rn4, see www.genome.ucsc.edu andhttp://genome.ucsc.edu/FAQ/FAQ.releases.html) unless otherwisespecified.

In one aspect the host non-human vertebrate mu constant region forforming the chimaeric IgM antibody/heavy chain may be at a different(non endogenous) chromosomal locus. In this case the inserted human DNA,ie, the human variable V,D,J and C gene segments, may then be insertedinto the non-human genome at a site which is distinct from that of thenaturally occurring heavy locus. Endogenous heavy chain expression isinactivated.

Reference to location of a human variable region, constant region or agene segment upstream or downstream of an endogenous mu constant regionmeans that there is a suitable relative location of the antibodynucleotide elements to enable formation of mu and then non-mu heavychains in vivo in the vertebrate. Thus, the inserted human DNA andendogenous constant region and any other endogenous sequences are inoperable connection with one another for antibody or antibody chainproduction.

As a source of human and non-human antibody gene segment sequences, theskilled person will also be aware of the following available databasesand resources (including updates thereof) the contents of which areincorporated herein by reference:

The Kabat Database (G. Johnson and T. T. Wu, 2002; World Wide Web (www)kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in 1966, theKabat database publishes aligned sequences of antibodies, T-cellreceptors, major histocompatibility complex (MHC) class I and IImolecules, and other proteins of immunological interest. A searchableinterface is provided by the Seqhuntll tool, and a range of utilities isavailable for sequence alignment, sequence subgroup classification, andthe generation of variability plots. See also Kabat, E. A., Wu, T. T.,Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteinsof Immunological Interest, 5th ed., NIH Publication No. 91-3242,Bethesda, Md., which is incorporated herein by reference, in particularwith reference to human gene segments for use in the present invention.

KabatMan (A. C. R. Martin, 2002; World Wide Web (www)bioinf.org.uk/abs/simkab.html). This is a web interface to make simplequeries to the Kabat sequence database.

IMGT (the International ImMunoGeneTics Information System®; M.-P.Lefranc, 2002; World Wide Web (www) imgt.cines.fr). IMGT is anintegrated information system that specializes in antibodies, T cellreceptors, and MHC molecules of all vertebrate species. It provides acommon portal to standardized data that include nucleotide and proteinsequences, oligonucleotide primers, gene maps, genetic polymorphisms,specificities, and two-dimensional (2D) and three-dimensional (3D)structures. IMGT includes three sequence databases (IMGT/LIGM-DB,IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3Dstructure database (IMGT/3Dstructure-DB), and a range of web resources(“IMGT Marie-Paule page”) and interactive tools.

V-BASE (I. M. Tomlinson, 2002; World Wide Web (www)mrc-cpe.cam.ac.uk/vbase). V-BASE is a comprehensive directory of allhuman antibody germline variable region sequences compiled from morethan one thousand published sequences. It includes a version of thealignment software DNAPLOT (developed by Hans-Helmar Althaus and WernerMüller) that allows the assignment of rearranged antibody V genes totheir closest germline gene segments.

Antibodies—Structure and Sequence (A. C. R. Martin, 2002; World Wide Web(www) bioinf.org.uk/abs). This page summarizes useful information onantibody structure and sequence. It provides a query interface to theKabat antibody sequence data, general information on antibodies, crystalstructures, and links to other antibody-related information. It alsodistributes an automated summary of all antibody structures deposited inthe Protein Databank (PDB). Of particular interest is a thoroughdescription and comparison of the various numbering schemes for antibodyvariable regions.

AAAAA (A Ho's Amazing Atlas of Antibody Anatomy; A. Honegger, 2001;World Wide Web (www) unizh.ch/^(˜)antibody). This resource includestools for structural analysis, modeling, and engineering. It adopts aunifying scheme for comprehensive structural alignment of antibody andT-cell-receptor sequences, and includes Excel macros for antibodyanalysis and graphical representation.

WAM (Web Antibody Modeling; N. Whitelegg and A. R. Rees, 2001; WorldWide Web (www) antibody.bath.ac.uk). Hosted by the Centre for ProteinAnalysis and Design at the University of Bath, United Kingdom. Based onthe AbM package (formerly marketed by Oxford Molecular) to construct 3Dmodels of antibody Fv sequences using a combination of establishedtheoretical methods, this site also includes the latest antibodystructural information.

Mike's Immunoglobulin Structure/Function Page (M. R. Clark, 2001; WorldWide Web (www) path.cam.ac.uk/^(˜)mrc7/mikeimages.html) These pagesprovide educational materials on immunoglobulin structure and function,and are illustrated by many colour images, models, and animations.Additional information is available on antibody humanization and MikeClark's Therapeutic Antibody Human Homology Project, which aims tocorrelate clinical efficacy and anti-immunoglobulin responses withvariable region sequences of therapeutic antibodies.

The Antibody Resource Page (The Antibody Resource Page, 2000; World WideWeb (www) antibodyresource.com). This site describes itself as the“complete guide to antibody research and suppliers.” Links to amino acidsequencing tools, nucleotide antibody sequencing tools, andhybridoma/cell-culture databases are provided.

Humanization by Design (J. Saldanha, 2000; World Wide Web (www)people.cryst.bbk.ac.uk/^(˜)ubcg07s). This resource provides an overviewon antibody humanization technology. The most useful feature is asearchable database (by sequence and text) of more than 40 publishedhumanized antibodies including information on design issues, frameworkchoice, framework back-mutations, and binding affinity of the humanizedconstructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo,Methods in Molecular Biology™, Human Press. Also at World Wide Web (www)blogsua.com/pdf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

Samples from which B-cells can be obtained include but are not limitedto blood, serum, spleen, splenic tissue, bone marrow, lymph, lymph node,thymus, and appendix. Antibodies and immunoglobulin chains can beobtained from each of the previous-mentioned samples and also from thefollowing non-limiting list of B-cells, ascites fluid, hybridomas, andcell cultures.

“Plurality” is used in the ordinary sense of the term and means “atleast one” or “more than one”.

“Derived from” is used in the ordinary sense of the term. Exemplarysynonyms include “produced as”, “resulting from”, “received from”,“obtained from”, “a product of”, “consequence of”, and “modified from”For example, a human variable region of a heavy chain can be derivedfrom recombination of human VH, D and JH gene segments and this reflectsthe in vivo recombination of these gene segments in, for example, atransgenic heavy chain locus according to the invention with anyaccompanying mutation (eg, junctional mutation). A constant region orgene segment sequence is derived from a human individual if, for exampleit is an identical nucleotide copy of that region or segment as found inthe genome of that individual (or found in a ethnic or geographicalpopulation of humans of which the individual is a member). Populationsand genomic variant immunoglobulin gene sequences can be found, eg, inthe 1000 Genomes database or by sampling DNA from humans and sequencing.

In one embodiment in any configuration of the invention, the genome of aor each vertebrate or cell has been modified to prevent or reduce theexpression of fully-endogenous antibody or heavy chains. Examples ofsuitable techniques for doing this can be found in WO2011004192, U.S.Pat. Nos. 7,501,552, 6,673,986, 6,130,364, WO2009/076464, EP1399559 andU.S. Pat. No. 6,586,251, the disclosures of which are incorporatedherein by reference. In one embodiment, the non-human vertebrate VDJregion of the endogenous heavy chain immunoglobulin locus, andoptionally VJ region of the endogenous light chain immunoglobulin loci(lambda and/or kappa loci), have been inactivated. For example, all orpart of the non-human vertebrate VDJ region is inactivated by inversionin the endogenous heavy chain immunoglobulin locus of the mammal,optionally with the inverted region being moved upstream or downstreamof the endogenous Ig locus. For example, all or part of the non-humanvertebrate VJ region is inactivated by inversion in the endogenous kappachain immunoglobulin locus of the mammal, optionally with the invertedregion being moved upstream or downstream of the endogenous Ig locus.For example, all or part of the non-human vertebrate VJ region isinactivated by inversion in the endogenous lambda chain immunoglobulinlocus of the mammal, optionally with the inverted region being movedupstream or downstream of the endogenous Ig locus. In one embodiment theendogenous heavy chain locus is inactivated in this way as is one orboth of the endogenous kappa and lambda loci.

Additionally or alternatively, the or each vertebrate has been generatedin a genetic background which prevents the production of mature host Band T lymphocytes, optionally a RAG-1-deficient and/or RAG-2 deficientbackground. See U.S. Pat. No. 5,859,301 for techniques of generatingRAG-1 deficient animals.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein.

An “isolated” antibody or heavy chain is one that has been identified,separated and/or recovered from a component of its productionenvironment (e.g., naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other components from itsproduction environment, eg, so that the antibody has been isolated to anFDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide, chain orantibody will be prepared by at least one purification step.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide, antigen, or epitope is one that binds to thatparticular polypeptide, antigen, or epitope without substantiallybinding to other polypeptides, antigens or epitopes. For example,binding to the antigen or epitope is specific when the antibody bindswith a K_(D) of 100 μM or less, 10 μM or less, 1 μM or less, 100 nM orless, eg, 10 nM or less, 1 nM or less, 500 pM or less, 100 pM or less,or 10 pM or less. The binding affinity (K_(D)) can be determined usingstandard procedures as will be known by the skilled person, eg, bindingin ELISA and/or affinity determination using surface plasmon resonance(eg, Biacore™ or KinExA™ solution phase affinity measurement which candetect down to fM affinities (Sapidyne Instruments, Idaho)).

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable carrier,excipient, or adjuvant” refers to an carrier, excipient, or adjuvantthat can be administered to a subject, together with an agent, e.g., anyantibody or antibody chain described herein, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the agent.

In one embodiment of any configuration of the vertebrate or cell of theinvention the genome comprises an antibody light chain locus whichcomprises all or part of the human Igλ locus including at least onehuman Jλ region and at least one human Cλ region, optionally C_(λ)6and/or C_(λ)7. Optionally, the light chain locus comprises a pluralityof human Jλ regions, optionally two or more of J_(λ)1, J_(λ)2, J_(λ)6and J_(κ)7, optionally all of J_(λ)1, J_(λ)2, J_(λ)6 and J_(λ)7. Thehuman lambda immunoglobulin locus comprises a unique gene architecturecomposed of serial J-C clusters. In order to take advantage of thisfeature, the invention in optional aspects employs one or more suchhuman J-C clusters. Thus, optionally the light chain locus comprises atleast one human J_(λ)-C_(λ) cluster, optionally at least J_(λ)7-C_(λ)7.The construction of such transgenes is facilitated by being able to useall or part of the human lambda locus such that the transgene comprisesone or more J-C clusters in germline configuration, advantageously alsoincluding intervening sequences between clusters and/or between adjacentJ and C regions in the human locus. This preserves any regulatoryelements within the intervening sequences which may be involved in VJand/or JC recombination and which may be recognised by endogenous AID(activation-induced deaminase).

An aspect provides a nucleotide sequence encoding the antibody or heavychain of the invention, optionally wherein the nucleotide sequence ispart of a vector. Suitable vectors will be readily apparent to theskilled person, eg, a conventional antibody expression vector comprisingthe nucleotide sequence together in operable linkage with one or moreexpression control elements.

An aspect provides a pharmaceutical composition comprising the antibodyor heavy chain of the invention and a diluent, excipient or carrier,optionally wherein the composition is contained in an IV container (eg,and IV bag) or a container connected to an IV syringe.

An aspect provides the use of the antibody or heavy chain of theinvention in the manufacture of a medicament for the treatment and/orprophylaxis of a disease or condition in a human patient.

The skilled person will generally be familiar with standard cloningtechniques and recombinant DNA technology—see e.g. Sambrook, J andRussell, D. (2001, 3^(rd) edition) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).

In a further aspect the invention relates to humanised antibodies andantibody heavy chains produced according to the present invention, anduse of said antibodies in human medicine. The invention also relates toa pharmaceutical composition comprising such an antibody or heavy chainand a pharmaceutically acceptable carrier or other excipient.

Methods for the generation of both monoclonal and polyclonal antibodiesare well known in the art, and the present invention relates to bothpolyclonal and monoclonal antibodies of fully humanised antibodies andheavy chains produced in response to antigen challenge in a vertebrate,cell or population of non-human vertebrates according to the presentinvention.

In a further aspect, the invention relates to use of a population ofnon-human vertebrates of the present invention in the analysis of thelikely effects of drugs and vaccines in the context of a human antibodyheavy chain repertoire.

The invention also relates to a method for identification or validationof a drug or vaccine, the method comprising delivering the vaccine ordrug to a population of vertebrates of the invention and monitoring oneor more of: the immune response, the safety profile; the effect ondisease.

The invention also relates to a kit comprising an antibody, heavy chainor antibody derivative as disclosed herein and either instructions foruse of such antibody, derivative or chain or a suitable laboratoryreagent, such as a buffer, antibody detection reagent.

The invention also relates to a method for making an antibody, or partthereof, the method comprising providing:

(i) a nucleic acid encoding an antibody or heavy chain, obtained usingthe vertebrate, cell or population of the present invention; or(ii) sequence information from which a nucleic acid encoding an antibodyor heavy chain, obtained using the vertebrate, cell or population of thepresent invention can be expressed to allow an antibody or heavy chainto be produced.

In an embodiment, each vertebrate is a non-human vertebrate, mouse orrat, whose genome comprises

(a) said gene locus, wherein the gene locus is a transgenic heavy chainlocus (ie, at the endogenous heavy chain locus position); and(b) an antibody kappa light chain locus transgene and/or an antibodylambda chain locus transgene;wherein all of the V, D and J in said transgenes are human V, D and J;wherein endogenous antibody heavy and light chain expression has beeninactivated; andoptionally wherein said genome is homozygous for said heavy and lightchain loci. This is useful for generating, predictably, only humanantibodies and chains in the vertebrate (and population comprising suchvertebrates) when all light chain constant regions are human.

In an embodiment, the kappa chain transgenic loci comprises asubstantially complete human functional Vκ and Jκ repertoire; and thelambda chain transgene comprises a substantially complete humanfunctional Vλ and Jλ repertoire. “Functional” here acknowledges that Iggene segment pseudogenes and non-functional human Ig gene segments canbe excluded.

In one example the gene locus of the invention is a heavy chain locuswhich comprises a S-mu switch (eg, endogenous S-mu) 5′ of the endogenousmu constant region and a human gamma constant region3′ of the endogenousmu constant region, with a S-gamma switch (eg, a human or endogenousS-gamma) between the Cmu and Cgamma regions. For example, the C-muregion and switches are mouse 129 C-mu region and switches; or the C-muregion and switches are mouse Black 6 C-mu region and switches. Inanother embodiment (where the non-human vertebrate species is mouse orrat), S-mu is a rat Smu and the C-mu region is mouse and optionallythere is a mouse S-gamma 3′ of the C-mu region.

In one aspect each vertebrate is a mouse whose genetic background isselected from mouse strains C57BL/6, M129 such as 129/SV, BALB/c, andany hybrid of C57BL/6, M129 such as 129/SV, or BALB/c. In an embodiment,each of these vertebrates have the same genetic background but two ormore of the vertebrates of the population differ in their human genesegment repertoires (eg, in their human C region gene segmentrepertoires).

In an embodiment, the J segments of each gene locus of the invention arehuman JH gene segments; optionally wherein each heavy chain locuscomprises a substantially complete functional repertoire of human JHgene segments.

In an embodiment, each gene locus of the invention comprises at least 2,3, 4, 5 or 6 different human JH gene segments.

In an embodiment, the D segments of each gene locus of the invention arehuman D gene segments; optionally wherein each heavy chain locuscomprises a substantially complete functional repertoire of human D genesegments.

In an embodiment, each gene locus of the invention comprises at least 5,10, 15, 20, 25, 26 or 27 different human D gene segments.

In an embodiment, the heavy chain loci of said vertebrates compriseidentical human D and JH gene segment repertoires, but differ in theirVH gene repertoires.

In an embodiment, each heavy chain locus comprises at least two human JHgene segments selected from the group consisting of J1, J2, J3, J4, J5and J6; optionally all of the gene segments of the group.

In an embodiment, each vertebrate comprises human VH gene segmentsselected from the group consisting of V6-1, V1-2, V1-3, V4-4, V7-41,V2-5, V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24,V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46,V3-48, V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69,V2-70, V3-72, V3-73 and V3- 74; wherein the VH gene repertoire comprisesa substantially complete human functional VH gene repertoire.

In an embodiment, endogenous antibody heavy chain expression has beeninactivated in the vertebrate or cell of the invention. For example,less than 10, 5, 4, 3, 2, 1 or 0.5% of heavy chains are provided byendogenous heavy chains (ie, heavy chains whose variable regions arederived from recombination of non-human vertebrate V, D and J genesegments).

The VL gene segments of a repertoire can, in one embodiment, berecombined VL, ie, provided as part of a variable region sequencederived from the recombination of human VL with JL (eg, where the VI andJL are human).

In an embodiment, the J segments of each transgenic light chain locusare human JL gene segments; optionally wherein each light chain locuscomprises a substantially complete functional repertoire of human Jκ orJλ gene segments (eg, each transgenic locus comprises human Vλ genesegments and Jλ gene segments, optionally a substantially completefunctional repertoire of human Jλ gene segments; or each transgeniclocus comprises human Vκ gene segments and Jκ gene segments, optionallya substantially complete functional repertoire of human Jκ genesegments).

In an embodiment, the kappa chain repertoire provided by said populationcomprises a substantially complete repertoire of functional human Vκgene segments; optionally providing at least 5 different human Jκ genesegments and at least at least 40 different human Vκ gene segments.

In an embodiment, the kappa chain repertoire provided by said populationcomprises at least 20, 25, 30, 35 or 40 different human Vκ genesegments.

In an embodiment, the lambda chain repertoire provided by saidpopulation comprises a substantially complete repertoire of functionalhuman Vλ gene segments; optionally providing at least 5 different humanJλ gene segments and at least 40 different human Vλ gene segments.

In an embodiment, the lambda chain repertoire provided by saidpopulation comprises at least 20, 25, 30, 35 or 40 different human Vλgene segments.

In an embodiment, each light chain locus of the vertebrate or cellcomprises at least 2, 3, 4, 5 or 6 different human Jκ or Jλ genesegments.

In an embodiment, the light chain loci of said vertebrate or cellcomprise identical human JL gene segment repertoires, but differ intheir VL gene repertoires.

In an embodiment, endogenous antibody kappa and/or lambda light chainexpression has been inactivated in the vertebrate or cell.

In an embodiment, said transgenic light chain loci of the vertebrate orcell are kappa light chain loci (at the endogenous kappa loci, ie,corresponding to the position of a kappa locus in a wild-type non-humanvertebrate genome). For example, a transgenic kappa locus can comprisehuman Vκ gene segments and Jκ gene segments upstream of a constantregion (eg, a CH, Cλ or Cκ gene segment; optionally which is anendogenous or human gene segment). For example, a transgenic kapppalocus can comprise human Vλ gene segments and Jλ gene segments upstreamof a constant region (eg, a CH, Cλ or Cκ gene segment; optionally whichis an endogenous or human gene segment).

In an embodiment, said transgenic light chain loci of the vertebrate orcell are lambda light chain loci (at the endogenous lambda loci, ie,corresponding to the position of a lambda locus in a wild-type non-humanvertebrate genome). For example, a transgenic lambda locus can comprisehuman Vκ gene segments and Jκ gene segments upstream of a constantregion (eg, a CH, Cλ or Cκ gene segment; optionally which is anendogenous or human gene segment). For example, a transgenic lambdalocus can comprise human Vλ gene segments and Jλ gene segments upstreamof a constant region (eg, a CH, Cλ or Cκ gene segment; optionally whichis an endogenous or human gene segment).

For selection of antibodies and heavy chains in the methods of theinvention, examples of a desirable antibody/chain characteristic areaffinity for binding a predetermined antigen or epitope (eg, asdetermined by surface plasmon resonance), completion with a knownantibody for binding to a predetermined antigen or epitope, epitopicspecificity of the antibody (eg, as determined by X-ray crystallography,competition with a known antibody for antigen binding wherein the knownantibody specifically binds to the antigen (eg, as determined by surfaceplasmon resonance, eg, Biacore™), performance in ELISA or anotherimmunoassay, a desirable biophysical characteristic (eg, meltingtemperature, pl, solution state, degree of aggregation, storage profileetc). In an embodiment, affinity is determined by surface plasmonresonance.

Methods of immunisation for use in the invention are well known to theskilled person and may involve a classic prime-boost regime, RIMMS orany other protocol. An adjuvant may be administered with the antigen, asis known in the art.

The invention provides a pharmaceutical composition comprising anantibody or heavy chain selected as described above or a derivativethereof that binds said antigen, together with a pharmaceuticallyacceptable diluent, carrier or excipient.

Examples of derivative antibodies/chains (according to any aspectherein) are antibodies/chains that have one or more mutations comparedto the isolated antibody or chain (eg, to improve antigen-bindingaffinity and/or to enhance or inactivate Fc function) Such mutantsspecifically bind the antigen. Mutation or adaptation to produce aderivative includes, eg, mutation to produce Fc enhancement orinactivation. A derivative can be an antibody following conjugation to atoxic payload or reporter or label or other active moiety.

In an embodiment of any configuration, the vertebrate is (or vertebratesof the populatio are) naïve (ie, not immunised with a predeterminedantigen, as the term is understood in the art; for example, such avertebrate that has been kept in a relatively sterile environment asprovided by an animal house used for R&D). In another example, thevertebrates have been immunised with a predetermined antigen, eg, anantigen bearing a human epitope. In another example, the populationcomprises naïve and immunised vertebrates.

In an embodiment of the population or method, the vertebrates have beenimmunised with the same antigen (eg, a human antigen).

In an embodiment of the population or method, the vertebrates are naïve.

In an embodiment of the population or method, the vertebrates have acommon collection of light chain loci. For example, the kappa chain locialleles are identical in the vertebrates and/or the lambda chain locialleles are identical in the vertebrates. This simplifies constructionof vertebrate variants for producing the population and also simplifiesbreeding.

In an embodiment, the method comprises comprising selecting one or moreantibody heavy chains (eg, as part of an antibodies) from saidpopulation or repertoire according to a desired characteristic (eg,affinity for biding an antigen).

In one example, the cell is a B-cell, hybridoma, ES cell or iPS cell. EScells and iPS cells can be used to develop corresponding non-humanvertebrates (vertebrates of the invention).

In an embodiment, the vertebrate species is selected from human, mouse,rat, rabbit, guinea pig, chicken, a fish, a bird, a reptile, a Camelid,bovine, chimpanzee, a non-human primate and a primate.

In an embodiment, the vertebrate is a mouse or rat.

Aspects of the invention are as follows:—

-   -   1. A non-human vertebrate (eg, a mouse or a rat) or a non-human        vertebrate cell (eg, a mouse cell or a rat cell) whose genome        comprises a gene locus for expression of antibody heavy chains,        the locus comprising        -   (a) an unrearranged human variable region comprising human            variable region gene segments for expression of a repertoire            of human variable domains;        -   (b) an endogenous mu constant region for expression of IgM            antibody heavy chains comprising endogenous mu heavy chain            constant domains and human variable domains; and        -   (c) a humanised non-mu constant region downstream of the mu            constant region for expression of non-mu antibody heavy            chains comprising human non-mu constant domains and human            variable domains;        -   Wherein the unrearranged variable region is provided as a            targeted insertion of the human variable region gene            segments upstream of the endogenous mu constant region in an            endogenous IgH locus such that the variable region gene            segments are able to recombine for expression and selection            in the context of an endogenous mu constant region.    -   2. The vertebrate or cell of aspect 1 comprising an antibody        heavy chain locus that comprises (in 5′ to 3′ direction) an        unrearranged human variable region, a first switch, an        endogenous mu constant region, a second switch and a human        constant region of said non-mu isotype; wherein the heavy chain        locus is capable of undergoing switching from IgM to the non-mu        isotype; optionally wherein the first switch is a rodent (eg,        mouse or rat) Smu or the endogenous Smu of the IgH locus.    -   3. The vertebrate or cell of aspect 1 or 2, wherein the genome        comprises endogenous RAG-1 and RAG-2 genes.    -   4. The vertebrate or cell of any preceding aspect, wherein the        human variable region comprises a plurality of unrearranged        human VH gene segments; optionally at least 5 different human VH        gene segments or a substantially complete human functional VH        gene repertoire.        -   For example, the variable region comprises human VH gene            segments selected from the group consisting of V6-1, V1-2,            V1-3, V4-4, V7-41, V2-5, V3-7, V1-8, V3-9, V3-11, V3-15,            V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30,            V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46, V3-48,            V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66,            V1-69, V2-70, V3-72, V3-73 and V3-74; wherein the VH gene            repertoire comprises a substantially complete human            functional VH gene repertoire.    -   5. The vertebrate or cell of any preceding aspect, wherein the        human variable region comprises a plurality of unrearranged        human D gene segments; optionally at least 5 or 20 different        human D gene segments or a substantially complete functional        repertoire of human D gene segments.        -   For example, the human variable region comprises at least 10            human D gene segments selected from the group consisting of            D1-1, D2-2, D3-3, D4-4, D5-5, D6-6, D1-7, D2-8, D3-9, D3-10,            D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18,            D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D1-26, D6-25 and            D7-27; optionally all of the gene segments of the group.    -   6. The vertebrate or cell of any preceding aspect, wherein the        human variable region comprises a plurality of unrearranged        human JH gene segments; at least 2 different human JH gene        segments or a substantially complete functional repertoire of        human D gene segments.    -   7. The vertebrate or cell of aspect 1 or 2, which does not        express endogenous antibody heavy chains of said non-mu isotype.    -   8. The vertebrate or cell of any preceding aspect, wherein the        non-mu constant region is a human gamma constant region.    -   9. The vertebrate or cell of aspect 8, wherein the gamma        constant region comprises CH2 and CH3 gene segments for encoding        an antibody Fc region.    -   10. The vertebrate or cell of aspect 9, wherein the CH2 and CH3        gene segments encode for an inactivated Fc region.    -   11. The vertebrate or cell of aspect 9, wherein the CH2 and CH3        gene segments encode for an activated Fc region.    -   12. The vertebrate or cell of any preceding aspect, wherein the        genome comprises no human Cmu genes.    -   13. The vertebrate or cell of any preceding aspect, wherein the        or all non-mu heavy chain constant regions in the genome        comprise a CH1.    -   14. The vertebrate or cell of any preceding aspect, wherein the        genome comprises a light chain locus comprising human VL and JL        gene segments upstream of a constant region for expression of        light chains comprising human variable regions; optionally        wherein the constant region is a human light chain constant        region.    -   15. The vertebrate or cell of aspect 14, which does not        substantially express endogenous light chains.    -   16. The vertebrate or cell of any one of aspects 8 to 11,        wherein the gamma constant region does not comprise a CH1 gene        segment.    -   17. The vertebrate or cell of aspect 16, wherein light chain        expression is inactive when the non-mu heavy chains are        expressed.    -   18. The vertebrate or cell of any preceding aspect, wherein the        locus comprises a replacement of endogenous non-mu constant        region gene segments (eg, CH2 and CH3 gene segments) with        corresponding human constant region gene segments (eg, CH2 and        CH3 gene segments) of the same non-mu isotype; optionally        wherein an endogenous switch of said non-mu isotype is retained.    -   19. The vertebrate or cell of any preceding aspect, wherein the        locus comprises an insertion of a human gamma constant region        nucleotide sequence comprising sequence from the human gamma CH2        gene segment to the human gamma CH3 gene segment, wherein the        insertion replaces a corresponding sequence of an endogenous        gamma constant region in the genome of the vertebrate or cell.    -   20. The vertebrate or cell of any preceding aspect, wherein        endogenous inter-gene segment sequences have been retained so        that the heavy chain locus comprises said human non-mu constant        region gene segments flanked by endogenous constant region        inter-gene segment sequences.    -   21. The vertebrate or cell of any preceding aspect, wherein the        locus comprises a replacement of endogenous gamma constant        region gene segments (eg, gamma CH2 and CH3 gene segments) with        human gamma constant region gene segments (eg, gamma CH2 and CH3        gene segments), but endogenous sequences between gamma constant        regions have been retained.    -   22. A population of vertebrates according to any preceding        aspect, wherein the population comprises        -   (i) a first vertebrate wherein the locus is an antibody            heavy chain locus that comprises one or more first human            non-mu C gene segment(s) (eg, a human CH1, CH2 or CH3; or an            Fc) or a first human constant region;        -   (ii) and a second vertebrate wherein the locus is an            antibody heavy chain locus that comprises one or more second            human non-mu C gene segment(s) (eg, a human CH1, CH2 or CH3;            or encoding an Fc) or a second constant region; and        -   wherein the first and second gene segments or regions are            the same type of C gene segment(s) (eg, both CH1 gene            segments or both encoding an Fc) or regions and the second            gene segment(s) is a variant of the first gene segment(s).    -   23. The population of aspect 22, wherein the first and second        constant regions are gamma constant regions of the same sub-type        (eg, both gamma-2 or gamma-3).    -   24. The population of aspect 22 or 23, wherein the second gene        segment is a synthetic mutant of the first gene segment.    -   25. The population of aspect 22, wherein one or both of the        first and second gene segment(s) encodes an inactivated human        gamma Fc.    -   26. The population of aspect 22, wherein one or both of the        first and second gene segment(s) encodes an activated human        gamma Fc.    -   27. A method of isolating an antibody or human heavy chain, the        method comprising immunising a vertebrate or population of        vertebrates according to any preceding aspect with an antigen        and isolating a non-mu antibody or heavy chain from said        vertebrate or a vertebrate of the population, wherein the        isolated antibody or heavy chain specifically binds to the        antigen and comprises (or is) a fully human heavy chain of said        non-mu isotype.    -   28. A pharmaceutical composition comprising the isolated        antibody or heavy chain recited in aspect 27 or a copy or        derivative thereof.    -   29. A method of obtaining a humanised and affinity matured        antigen-specific antibody heavy chain, the method comprising        humanising the heavy chain in vivo in a non-human vertebrate        (eg, a mouse or a rat) comprising functional RAG and activation        induced cytidine deaminase (AID) by immunising the vertebrate        with the antigen and obtaining recombination of VH,D and JH gene        segments in vivo, somatic hypermutation and isotype switching in        a B-cell of the vertebrate from an endogenous mu isotype to a        human non-mu isotype, wherein a repertoire of affinity matured        antigen-specific non-mu antibody heavy chains are produced and        expressed by the vertebrate, the non-mu constant domains of the        heavy chains being human constant domains, the method further        comprising isolating one or more of said humanised heavy chains.    -   30. The method of aspect 29, wherein the variable domain of each        isolated heavy chain is a human variable domain.    -   31. The method of aspect 29 or 30, wherein the vertebrate is        according to any one of aspects 1 to 21.    -   32. A pharmaceutical composition comprising an isolated antibody        heavy chain obtained in the method of aspect 29, 30 or 31 or a        copy or derivative thereof; optionally wherein the heavy chain,        copy or derivative is provided by an antibody that specifically        binds the antigen.    -   33. The method of any one of aspects 27, 29, 30 or 31 comprising        the step of isolating a B-cell from said immunised vertebrate        (or an immunised vertebrate of said population), wherein the        B-cell expresses a said isolated antibody or heavy chain; and        optionally immortalising the B-cell.    -   34. The method of any one of aspects 27, 29, 30, 31 or 32,        comprising isolating a nucleotide sequence from said immunised        vertebrate or B-cell, wherein the nucleotide sequence encodes a        said isolated antibody or a heavy chain thereof.    -   35. A vector (optionally in a host cell) comprising the        nucleotide sequence recited in aspect 34 or a copy thereof.    -   36. A method of treating or preventing a medical condition or        disease in a human associated or caused by said antigen, the        method comprising administering to the human the        antigen-specific antibody or heavy chain obtained in the method        of any one of aspects 21, 23, 24, 25, 27 and 28.    -   37. The antigen-specific antibody or heavy chain obtained in the        method of any one of aspects 27, 29, 30 or 31 for use in the        treatment or prophylaxis of a medical condition or disease in a        human associated or caused by said antigen.    -   38. Use of the antigen-specific antibody or heavy chain obtained        in the method of any one of aspects 27, 29, 30 or 31 in the        manufacture of a medicament for use in the treatment or        prophylaxis of a medical condition or disease in a human        associated or caused by said antigen.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention and(unless the context states otherwise) embodiments can be applied to anyof the configurations of the invention described herein. The principalfeatures of this invention can be employed in various embodimentswithout departing from the scope of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routinestudy, numerous equivalents to the specific procedures described herein.Such equivalents are considered to be within the scope of this inventionand are covered by the claims. All publications and patent applicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term “or” in the claims is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variationof error for the device, the method being employed to determine thevalue, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions, populations, vertebrates, antibodies,repertoires and/or methods disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting prophetic Examples.

EXAMPLES Example 1 “Recombineered BAC Vectors to Add Human C GeneSegments to the Mouse Genome”

Methods of replacing endogenous antibody gene segments withcorresponding human gene segments harboured by BACs are generally knownin the art, eg, as disclosed in WO02066630, WO2011163311 & WO2011163314(Regeneron). These methods use standard homologous recombination. As analternative, recombinase mediated cassette exchange (RMCE; seeWO2011004192 & WO2011158009 (Kymab Limited)) can be used withcre-lox-mediated, transposon-mediated (eg, using piggyBac) or homologousrecombination-mediated deletion of endogenous non-mu constant regionsequences, in order to replace endogenous non-mu with human constantregion gene segments.

Modified BACs with human C gene segments, for example created using themethods described in Example 1, can be used to alter the genome ofnon-human mammals (eg, mice or rats). These alterations can result in anintact Ig heavy chain locus in which normal immunoglobulin gene segmentrecombination results in VDJ combinations which includes human VH, D andJH gene segments. An example of how to insert human V, D and J genesegments into a non-human vertebrate genome is disclosed in theRegeneron and Kymab PCT applications cited above. These methods are alsouseful for inserting human C region sequences.

Standard recombineering methods can be used to introduce human C genesegments and regions into BACs for use in inserting non-human vertebrategenomic DNA into the genome of a non-human vertebrate embryonic stemcell (ES cell), eg, using standard homologous recombination or RMCE. Inone embodiment, as a first step a genomic fragment containing a humanconstant region nucleotide sequence (encoding one or more human constantdomains) of a human genomic sequence is inserted into a bacterialartificial chromosome (BAC) vector by standard techniques. Preferably,such a BAC, which can range in size from 20-kb to 200-kb or more, can beisolated from commercially available libraries of BACs (eg, Caltec A, B,C or D human BAC library or RPCI-11 human BAC library, Invitrogen) bystandard techniques including sequence searches or by hybridization tobacterial colonies containing BACs to identify those with a BAC ofinterest.

In the present example, a BAC is chosen that contains a human gamma-1constant region nucleotide sequence comprising from the CH1 gene segmentto the polyA immediately 5′ of M2 of a human genomic sequence. Usingconventional techniques, the endogenous gamma-1 constant region genesegments can be replaced by the corresponding human gamma-1 constantregion gene segments. In this example, two steps are required. The firststep replaces the C domain coding exon of an endogenous C gene segmentwith a positive-negative selection operon, in this example, an operonencoding an ampicillin resistance gene (Amp) and astreptomycin-sensitizing ribosomal protein (rpsL). Certain strains ofbacteria can be selected for the absence of the rpsL gene by resistanceto streptomycin. Short stretches of DNA homologous to sequences flankingthe endogenous C gene exon are placed 5′ and 3′ of the rpsL-Amp operon.In the presence of appropriate recombination factors per standardrecombineering techniques (eg, homologous recombination) recombinationbetween the operon fragment and the BAC will result in replacement ofthe endogenous C gene exon with the operon which are selected byresistance to ampicillin. The second step uses the same homologoussequences in order to replace the inserted operon with a correspondinghuman C gene segment. Successful integrations of the human gene segmentare selected in bacteria that become resistant to streptomycin due tothe loss of the operon, specifically the rpsL portion.

In this example, the two step process as described can be repeated foreach of the endogenous C gene segments until the desired endogenousnon-mu constant region has been humanised by replacement of endogenousconstant region gene segments with human counterparts. The method can beused similarly to replace an entire endogenous non-mu constant region(including gene segments and inter-gene segment sequences) with thecorresponding human non-mu constant region sequence; or just theendogenous C gene segments of the non-mu constant region can be placed(leaving endogenous inter-gene segment sequences in place between thehuman C gene segments).

As is standard, genetically-manipulated stem cells can be inserted intoblastocysts and implanted into foster mothers in order to give rise tolive-born progeny animals bearing human C gene segments in heavy chainloci according to the invention. Standard breeding can yield progenythat are homozygous for the heavy chain locus of the invention and inwhich endogenous heavy chain expression (ie, chains comprisingendogenous variable regions) has been inactivated using conventionaltechniques.

Example 2 “Adding Human C Gene Segments to a Non-Human Vertebrate GenomeUsing SRMCE of Modified BACs”

As mentioned above, one technique to integrate modified BACs with humanC gene segments into a genome is sequential recombinase mediatedcassette exchange (sRMCE). The technique is described in WO2011004192(Kymab Limited), which is incorporated here in its entirety byreference.

sRMCE provides for a locus modified with a ‘landing pad’ inserted at aspecific location. This insertion can either be de novo via homologousrecombination or as a consequence of a previous BAC insertion. In thisexample, the landing pad is inserted in the mouse IgH locus (in a mouseES cell) 3′ of the mouse C-mu region. In the present example, theinsertion is between the mouse C-mu and mouse C-gamma regions (eg,between mouse Cmu and Cdelta or between mouse Cdelta and mouse gamma-1).Using standard sRMCE with serial BACs, a human gamma-1 constant regionis built up in the genome of the ES cell (and optionally the mousegamma-1 constant region or all mouse non-mu constant regions are deletedor replaced similarly with the corresponding human constant regionsequences). In another example, serial BACs are used with sRMCE toinsert the entire human sequence from the human delta or gamma-3constant region to (and including) the human gamma A2 constant region.The mouse constant region from delta or gamma-1 to (and including) alphais deleted (or just the mouse gamma constant regions are deleted). BACinsertion via SRMCE techniques are also used for the addition of humanVH, DH and J gene segments immediately downstream of the 3′-most mouseJH gene segment. Endogenous mouse VDJ is inactivated or deleted.

An alternative embodiment is to replace the endogenous mouse C gamma-1with a ‘landing pad’ via homologous recombination. Using standard sRMCEa construct containing human C gamma-1 exons 1 to 4, fused to mouse Cgamma-1 transmembrane and cytoplasmic exons, replaces the endogenous Cgamma-1 segments. The construct is assembled by PCR from human and mouseBACS exploiting a conserved SPGK amino acid motif at the 3′ end of exon4 in both human and mouse C gamma-1, which aids construction using PCR.The rationale for using a chimeric C gamma-1 construct is that thesecretory form of IgG1 is fully human whilst the membrane-bound form ofIgG1 maintains normal mouse signalling.

A further embodiment would be to use the same landing pad and a similarhuman-mouse C gamma-1 construct described previously, but in thisexample the construct lacks human C gamma-1 exon 1 and so ablates heavyand light chain pairing, thus a heavy-chain only IgG1 is expressed.

ES cell clones with correct insertions are selected from a pool ofclones without insertions or with non-productive insertions byresistance to puromycin as described in WO2011004192. Resistance topuromycin results from the juxtaposition of an active promoter element,PGK, with the puroTK coding region. Correct insertions are verified bystandard techniques including PCR of junctions, PCR of internalelements, Southern blotting, comparative genomic hybridization (CGH),sequencing and etc. In the example, as described in WO2011004192,correct lox2272-lox2272 and loxP-loxP recombination also results in twointact sets of piggyBac elements that did not exist prior to insertion.An intact piggyBac element is comprised of a set of inverted repeatswhich are depicted in the figure by “PB5′” and “PB3′”. An appropriatedoriented set of piggyBac elements are the substrate of piggyBactransposase which can catalyse recombination between the elements,resulting in deletion of intervening sequences as well as both elements.The DNA remaining after a piggyBac transposition is left intact and islacking any remnant of the piggyBac element. In the example, ES cellclones with successful piggyBac transposition are selected by loss ofthe active puroTK element which renders the cells resistant to the drugFIAU.

In an alternative method, it is possible to use standard homologousrecombination using BACs instead of RMCE (eg, see the techniquesdisclosed in the Regeneron and Ablexis PCT applications mentionedabove). In this case, vectors would be constructed in which each vectorcomprises a stretch of genomic human constant region DNA flanked byhomology arms. The homology arms would have the sequence of the DNA inthe recipient ES cell genome that flanks the insertion site (forexample, flanking a mouse constant region sequence to be replaced byinsertion of the human DNA). Using serial homologous recombination steps(optionally using BACS with overlapping homology arms as described inWO2009076464) the human constant region(s) can be built up in a processthat replaces mouse constant region DNA. Alternatively, insertion ofhuman DNA can be in a separate step from deletion of mouse constantregion DNA (eg, using site-specific recombination such as cre-lox ortransposon-mediated deletion such as using piggyBac).

The final product is an ES cell whose genome comprises a transgenic IgHlocus with a plurality of human VH, D and JH gene segments upstream of amouse S-mu switch, mouse mu constant region, a gamma switch (optionallythe mouse S-gamma is retained, or the human S-gamma can be insertedtogether with the other human gamma constant region sequences) and ahuman gamma constant region (eg, at least a human gamma-1 constantregion). Expression of mouse variable regions is inactivated (eg, byinversion of the mouse V, D, J as described in WO2011004192 or bydeletion of all or part (eg, the J gene segments) of the mouse IgHvariable region gene segments. Using standard techniques progeny miceare developed from the transgenic ES cells (the mice being preferablyhomozygous for the transgenic heavy chain locus). As is well known tothose skilled in the art, an ES cell clone can be used to create a lineof genetically modified mice via injection of said cells into a mouseblastocyst embryo, transferring the injected embryo to a suitablerecipient and breeding the chimeric offspring that result. The modifiedgene locus can be propagated through breeding and made eitherheterozygous or homozygous depending on the genetic cross.

The human variable region gene segments are positioned such that theycan participate in the recombination events associated with B cellmaturation yielding VDJ gene segments and mouse C-mu constant domains.Isotype switching to a human constant region (eg, C gamma-1) will yieldtranscripts that encode fully human, mature heavy chains comprisinghuman variable regions (with somatic hypermutation caused by mouse AID)and human constant domains. Productive transcripts will be expressed bythe progeny mice (ie, the mouse's in vivo antibody-producing machineryis harnessed to select for productive transcripts that can be expressed)and the skilled person will know at the outset that all gamma-1 typechains will be human (where the heavy chain loci have been humanised toinclude human gamma-1 constant region and exclude mouse gamma-1 constantregion). Commercially-available antibodies can be used to select andisolated specific human IgG isotypes (eg, rabbit anti-human IgG1,Catalogue Number: AP20578PU-N from Acris Antibodies GmbH; mouseanti-human IgG2, Catalogue Number: AM08152AP-N from Acris AntibodiesGmbH; mouse anti-human IgG3, Catalogue Number: MCA516P or sheep-antihuman Ig3, Catalogue Number: AP05355HR-N from AbD Serotec; mouseanti-human IgG4, Catalog NumberMCA2098P from AbD Serotec). For example,progeny mice are immunised with a human target antigen or a viral orbacterial antigen and IgG1 antibodies are selected for antigen bindingisolated. These will have fully human heavy chains and be specific forthe target antigen. If the progeny mice are engineered (using standardtechniques, eg, as disclosed in the Kymab Limited or Regeneron PCTapplications disclosed above) to include humanised light chain loci (eg,kappa and/or lambda light chain loci in which the mouse constant andvariable regions have been deleted or inactivated and there has beeninserted human light chain variable region VL and JL gene segments andoptionally also human constant regions) the isolated IgG1 antibodieswill have fully human variable region and, if the light chain loci bearonly human constant region genes, the IgG1 antibodies will be fullyhuman, well expressed and selected by an in vivo system. Additionally,they will specifically bind target antigen when the mice have beenimmunised and IgG1 have been selected against said target. Thisrepresents a reliable and much simpler technique for obtaining fullyhuman antibodies (or antibodies with fully human heavy chains) than aspresently possible with the multi-step methods of the art which rely onin vitro manipulation and selection using antibody engineering tohumanise antibodies.

In a variation, light chain expression is inactivated in progeny mice,eg, by deletion of the mouse kappa (and optionally also lambda) variableregion or part thereof (eg, JL gene segments) or by deletion of kappa(and optionally also lambda) constant regions in the light chain loci.In this case, human gamma constant regions are inserted (eg, for all IgGsubtypes, or at least for IgG1), in which the human CH1 gene segments(and optionally hinge regions) are omitted. In the mouse mu constantregion, CH1 can be retained (eg, see WO2011072204, the disclosure ofwhich is incorporated herein by reference). These mice produceheavy-chain only antibodies (H2 antibodies) lacking CH1 domains anddevoid of light chains. These heavy-chain only antibodies are fullyhuman (human variable and constant domains, eg human gamma-1 constantdomains when the heavy chain loci in the mice have been humanised byinsertion of human gamma-1 constant regions and inactivation or deletionof mouse gamma-1 constant regions). Following immmunisation with a humantarget antigen or a viral or bacterial antigen, heavy chain-onlyantibodies can be selected that are specific for the antigen ofinterest, fully human, well expressed and selected by an in vivo system.

I claim:
 1. A method of obtaining an antigen-specific antibody orantigen binding fragment thereof, said antibody comprising a humanimmunoglobulin heavy (IgH) chain comprising a human IgH chain variableregion and a human IgH chain constant region, said antigen bindingfragment comprising said human IgH chain variable region, the methodcomprising: expressing said antibody or antigen binding fragment thereoffrom a cell comprising nucleic acid encoding said human IgH chainvariable region of said antibody, wherein said human IgH chain variableregion is of a mouse whose genome comprises a gene locus for expressionof antibody heavy chains, said locus comprising: (a) an unrearrangedhuman heavy chain variable region comprising human heavy chain variableregion gene segments comprising a plurality of unrearranged humanvariable gene segments (VH), one or more unrearranged human D genesegments (D) and one or more human JH gene segments (JH) for expressionof a plurality of human heavy chain variable domains; and (b) a heavychain constant region comprising one or more human gamma constantregions selected from the group consisting of gamma-1, gamma-2, gamma-3and gamma-4, wherein each of said one or more human gamma constantregions comprises CH2, CH3, M1 and M2 gene segments, said (a) and (b)encoding gamma antibody heavy chains comprising (i) gamma heavy chainconstant domains and (ii) human heavy chain variable domains; whereinsaid unrearranged human heavy chain variable region is upstream of andoperably linked to said gamma heavy chain constant region and comprisedby an IgH locus, wherein one or more of said human CH2 and human CH3gene segments are in place of the respective CH2 and CH3 gene segmentsof endogenous gamma heavy chain constant region DNA, such that saidhuman heavy chain variable region gene segments are able to recombinefor expression of a gamma antibody heavy chain comprising a human gammaheavy chain antibody Fc region comprising a human CH2 and a human CH3and a human heavy chain variable domain.
 2. The method according toclaim 1, wherein said genome of said mouse comprises endogenousrecombination activating gene 1 (RAG-1) and recombination activatinggene 2 (RAG-2) genes.
 3. The method according to claim 1, wherein saidunrearranged human variable region of said locus of said mouse comprisesa plurality of unrearranged human D and/or JH gene segments.
 4. Themethod according to claim 3, wherein said plurality of unrearrangedhuman D gene segments of said mouse comprises at least 5 different humanD gene segments.
 5. The method according to claim 4, wherein saidplurality of unrearranged human JH gene segments of said mouse,comprises at least 2 different human JH gene segments.
 6. The methodaccording to claim 1, wherein said human gamma CH2 and human gamma CH3gene segments of said mouse encode an antibody Fc region.
 7. The methodaccording to claim 6, wherein said human gamma CH2 and human gamma CH3gene segments of said mouse encode an inactive Fc region.
 8. The methodaccording to claim 6, wherein said human gamma CH2 and human gamma CH3gene segments of said mouse encode an active Fc region.
 9. The methodaccording to claim 6, wherein said genome of said mouse comprises nohuman Cμ genes.
 10. The method according to claim 1, wherein saidgamma-1 constant region of said mouse comprises a human CH1 genesegment.
 11. The method according to claim 6, wherein said gamma-1constant region of said mouse does not comprise a human CH1 genesegment.
 12. The method according to claim 11, wherein light chainexpression is inactive in said mouse when non-mu heavy chains areexpressed in the mouse.
 13. The method according to claim 12, whereinsaid IgH locus comprises said human CH2 and human CH3 gene segmentswhich are in place of corresponding endogenous non-mu heavy chainconstant region gene segments of the same non-mu isotype.
 14. The methodaccording to claim 6, wherein said human gamma CH2 and human gamma CH3gene segments are in place of endogenous gamma constant region DNA insaid genome of said mouse, whereby the human gamma CH2 and CH3 genesegments are functional to encode a gamma antibody Fc region.
 15. Themethod according to claim 1, wherein said heavy chain constant regioncomprising one or more human gamma constant regions of said mousecomprises a human gamma-1 constant region comprising human CH1, humanCH2 and human CH3.
 16. The method according to of claim 15, wherein saidendogenous IgH locus of said mouse does not comprise a mouse gamma-1constant region.
 17. The method according to claim 1, wherein said mousedoes not express endogenous antibody heavy chains of a non-mu isotype.18. The method according to claim 6, wherein said human gamma constantregion of said mouse comprises a human CH1 gene segment.
 19. The methodaccording to claim 1, wherein said human gamma constant region of saidmouse does not comprise a human CH1 gene segment.
 20. The methodaccording to claim 1, wherein said human CH2 and human CH3 gene segmentsof said mouse are human CH2 gamma-1 and human CH3 gamma-1 gene segments.21. The method according to claim 6, wherein said human CH2 and humanCH3 gene segments of said mouse are human CH2 gamma-1 and human CH3gamma-1 gene segments.
 22. The method according to claim 6, wherein saidhuman gamma constant region of said mouse comprises a human gamma CH1gene segment in place of endogenous mouse gamma CH1 gene segment. 23.The method according to claim 1, wherein said mouse is homozygous forinsertion of the human DNA in first and second copies of an IgH antibodylocus.
 24. The method of claim 1, wherein said one or more human gammaconstant regions of said mouse further comprise the poly-A immediately3′ of CH3 or M2.
 25. A method of obtaining an antigen-specific antibodycomprising a human immunoglobulin heavy (IgH) chain, a human antibodyheavy (IgH) chain or antigen binding fragment thereof, said heavy (IgH)chain comprising a human IgH chain variable region and a human IgH chainconstant region, said antigen binding fragment comprising said human IgHchain variable region, the method comprising: expressing said antibody,heavy chain or antigen binding fragment thereof from a cell comprisingnucleic acid encoding said human IgH chain variable region of saidantibody, wherein said human IgH chain variable region is of atransgenic mouse contacted with said antigen, said mouse comprising inits germline a chimeric homozygous immunoglobulin heavy chain locus;wherein the germline of said mouse comprises a gene locus for expressionof antibody heavy chains, said locus comprising: (a) an unrearrangedhuman heavy chain variable region comprising human heavy chain variableregion gene segments comprising a plurality of unrearranged humanvariable gene segments (VH), one or more unrearranged human D genesegments (D) and one or more human JH gene segments (JH) for expressionof a plurality of human heavy chain variable domains; and (b) a non-muheavy chain constant region comprising non-mu heavy chain constantregion gene segments comprising a human CH2 gene segment and a human CH3gene segment encoding non-mu antibody heavy chains comprising (i) non-muheavy chain constant domains and (ii) human heavy chain variabledomains; wherein said unrearranged human heavy chain variable region ofsaid mouse is upstream of and operably linked to said non-mu heavy chainconstant region and comprised by an IgH locus and said human CH2 andhuman CH3 gene segments are in place of endogenous non-mu heavy chainconstant region DNA, such that said human heavy chain variable regiongene segments are able to recombine for expression of a non-mu antibodyheavy chain comprising a human non-mu heavy chain antibody Fc regioncomprising a human CH2 and a human CH3 and a human heavy chain variabledomain, wherein in said genome, human DNA is incorporated in place ofendogenous Ig antibody loci DNA, “and wherein said human variable regiongene segments are positioned to recombine to human VDJ gene segments andwould be operably linked to endogenous mouse constant region if present.26. The method according to claim 25, wherein said genome of said mousecomprises endogenous recombination activating gene 1 (RAG-1) andrecombination activating gene 2 (RAG-2) genes.
 27. The method accordingto claim 25, wherein said unrearranged human variable region of saidlocus of said mouse comprises a plurality of unrearranged human D and/orJH gene segments.
 28. The method according to claim 27, wherein saidplurality of unrearranged human D gene segments of said mouse comprisesat least 5 different human D gene segments.
 29. The method according toclaim 28, wherein said plurality of unrearranged human JH gene segmentsof said mouse, comprises at least 2 different human JH gene segments.30. The method according to claim 25, wherein said non-mu constantregion of said locus of said mouse comprises a human gamma constantregion comprising a human gamma CH2 gene segment and a human gamma CH3gene segment, wherein said human gamma CH2 and human gamma CH3 genesegments encode an antibody Fc region.
 31. The method according to claim30, wherein said CH2 and CH3 gene segments of said mouse encode aninactive Fc region.
 32. The method according to claim 30, wherein saidCH2 and CH3 gene segments of said mouse encode an active Fc region. 33.The method according to claim 30, wherein said genome of said mousecomprises no human Cμ genes.
 34. The method according to claim 25,wherein said non-mu heavy chain constant region of said mouse comprisesa human CH1 gene segment.
 35. The method according to claim 30, whereinsaid gamma constant region of said mouse does not comprise a human CH1gene segment.
 36. The method according to claim 35, wherein light chainexpression is inactive in said mouse when non-mu heavy chains areexpressed in the mouse.
 37. The method according to claim 36, whereinsaid IgH locus comprises said human CH2 and human CH3 gene segments arein place of corresponding endogenous non-mu heavy chain constant regiongene segments of the same non-mu isotype.
 38. The method according toclaim 30, wherein said human gamma CH2 and human gamma CH3 gene segmentsare in place of endogenous gamma constant region DNA in said genome ofsaid mouse, whereby the human gamma CH2 and CH3 gene segments arefunctional to encode a gamma antibody Fc region.
 39. The methodaccording to claim 25, wherein said human non-mu constant region of saidmouse comprises a human gamma-1 constant region comprising human CH1,human CH2 and human CH3.
 40. The method according to of claim 39,wherein said mouse does not comprise a mouse gamma-1 constant region.41. The method according to claim 25, wherein said mouse does notexpress endogenous antibody heavy chains of said non-mu isotype.
 42. Themethod according to claim 30, wherein said human gamma constant regionof said mouse comprises a human CH1 gene segment.
 43. The methodaccording to claim 25, wherein said non-mu heavy chain constant regionof said mouse does not comprise a human CH1 gene segment.
 44. The methodaccording to claim 25, wherein said human CH2 and human CH3 genesegments of said mouse are human CH2 gamma-1 and human CH3 gamma-1 genesegments.
 45. The method according to claim 30, wherein said human CH2and human CH3 gene segments of said mouse are human CH2 gamma-1 andhuman CH3 gamma-1 gene segments.
 46. The method according to claim 30,wherein said human non-mu constant region of said mouse comprises ahuman gamma CH1 gene segment in place of endogenous mouse gamma CH1 genesegment.
 47. The method according to claim 25, wherein said mouse ishomozygous for insertion of the human DNA in first and second copies ofan endogenous IgH antibody locus.
 48. A method of obtaining a nucleicacid encoding a human antibody heavy (IgH) chain variable region, themethod comprising: obtaining or copying a nucleic acid encoding saidhuman IgH chain variable region of said antibody, wherein said human IgHchain variable region is of a transgenic mouse contacted with saidantigen, said mouse comprising in its germline a chimeric homozygousimmunoglobulin heavy chain locus; wherein the germline of said mousecomprises a gene locus for expression of antibody heavy chains, saidlocus comprising: (a) an unrearranged human heavy chain variable regioncomprising human heavy chain variable region gene segments comprising aplurality of unrearranged human variable gene segments (VH), one or moreunrearranged human D gene segments (D) and one or more human JH genesegments (JH) for expression of a plurality of human heavy chainvariable domains; and (b) a non-mu heavy chain constant regioncomprising non-mu heavy chain constant region gene segments comprising ahuman CH2 gene segment and a human CH3 gene segment encoding non-muantibody heavy chains comprising (i) non-mu heavy chain constant domainsand (ii) human heavy chain variable domains; wherein said unrearrangedhuman heavy chain variable region of said mouse is upstream of andoperably linked to said non-mu heavy chain constant region and comprisedby an IgH locus, and wherein said human CH2 and human CH3 gene segmentsare in place of endogenous non-mu heavy chain constant region DNA, suchthat said human heavy chain variable region gene segments are able torecombine for expression of a non-mu antibody heavy chain comprising ahuman non-mu heavy chain antibody Fc region comprising a human CH2 and ahuman CH3 and a human heavy chain variable domain, wherein in saidgenome, human DNA is only in place of endogenous Ig antibody loci DNA.49. The method according to claim 48, wherein said genome of said mousecomprises endogenous recombination activating gene 1 (RAG-1) andrecombination activating gene 2 (RAG-2) genes.
 50. The method accordingto claim 48, wherein said unrearranged human variable region of saidlocus of said mouse comprises a plurality of unrearranged human D and/orJH gene segments.
 51. The method according to claim 50, wherein saidplurality of unrearranged human D gene segments of said mouse comprisesat least 5 different human D gene segments.
 52. The method according toclaim 51, wherein said plurality of unrearranged human JH gene segmentsof said mouse, comprises at least 2 different human JH gene segments.53. The method according to claim 48, wherein said non-mu constantregion of said locus of said mouse comprises a human gamma constantregion comprising a human gamma CH2 gene segment and a human gamma CH3gene segment, wherein said human gamma CH2 and human gamma CH3 genesegments encode an antibody Fc region.
 54. The method according to claim53, wherein said CH2 and CH3 gene segments of said mouse encode aninactive Fc region.
 55. The method according to claim 53, wherein saidCH2 and CH3 gene segments of said mouse encode an active Fc region. 56.The method according to claim 53, wherein said genome of said mousecomprises no human Cμ genes.
 57. The method according to claim 48,wherein said non-mu heavy chain constant region of said mouse comprisesa human CH1 gene segment.
 58. The method according to claim 53, whereinsaid gamma constant region of said mouse does not comprise a human CH1gene segment.
 59. The method according to claim 58, wherein light chainexpression is inactive in said mouse when non-mu heavy chains areexpressed in the mouse.
 60. The method according to claim 59, whereinsaid IgH locus comprises said human CH2 and human CH3 gene segments arein place of corresponding endogenous non-mu heavy chain constant regiongene segments of the same non-mu isotype.
 61. The method according toclaim 53, wherein said human gamma CH2 and human gamma CH3 gene segmentsare in place of endogenous gamma constant region DNA in said genome ofsaid mouse, whereby the human gamma CH2 and CH3 gene segments arefunctional to encode a gamma antibody Fc region.
 62. The methodaccording to claim 48, wherein said human non-mu constant region of saidmouse comprises a human gamma-1 constant region comprising human CH1,human CH2 and human CH3.
 63. The method according to of claim 62,wherein said endogenous IgH locus of said mouse does not comprise amouse gamma-1 constant region.
 64. The method according to claim 48,wherein said mouse does not express endogenous antibody heavy chains ofsaid non-mu isotype.
 65. The method according to claim 53, wherein saidhuman gamma constant region of said mouse comprises a human CH1 genesegment.
 66. The method according to claim 48, wherein said non-mu heavychain constant region of said mouse does not comprise a human CH1 genesegment.
 67. The method according to claim 48, wherein said human CH2and human CH3 gene segments of said mouse are human CH2 gamma-1 andhuman CH3 gamma-1 gene segments.
 68. The method according to claim 53,wherein said human CH2 and human CH3 gene segments of said mouse arehuman CH2 gamma-1 and human CH3 gamma-1 gene segments.
 69. The methodaccording to claim 53, wherein said human non-mu constant region of saidmouse comprises a human gamma CH1 gene segment in place of endogenousmouse gamma CH1 gene segment.
 70. The method according to claim 48,wherein said mouse is homozygous for insertion of the human DNA in firstand second copies of an endogenous IgH antibody locus.
 71. The method ofclaim 48, wherein in said method of obtaining a nucleic acid encoding ahuman IgH chain variable region, said nucleic acid encoding said humanIgH chain variable region is upstream of a nucleotide sequence encodinga human constant region.
 72. The method of claim 71, further comprisingexpressing a human IgH chain, where said human IgH chain is expressed bysaid nucleic acid encoding said human IgH chain variable region upstreamof said nucleotide sequence encoding said human constant region.
 73. Themethod of claim 72, further comprising isolating (i) said human IgHchain expressed by said nucleic acid encoding said human IgH chainvariable region upstream of said nucleotide sequence encoding said humanconstant region or (ii) an antibody comprising said human IgH chain of(i).
 74. The method of claim 73, further comprising formulating apharmaceutical composition comprising said isolated human IgH chain oran antibody comprising said human IgH chain.
 75. The method of claim 71,further comprising inserting said nucleic acid encoding said human IgHchain variable region upstream of a nucleotide sequence encoding a humanconstant region into a vector.
 76. The method of claim 48, furthercomprising inserting said nucleic acid encoding said human IgH chainvariable region into a vector.
 77. The method of claim 76, wherein saidvector is an expression vector.
 78. The method of claim 76, furthercomprising inserting the vector of claim 93 into a host cell.
 79. Themethod of claim 77, further comprising inserting the expression vectorof claim 94 into a host cell.
 80. The method of claim 1, wherein saidhuman gamma-1 constant region of said mouse comprises a human IGHG1*01CH2 gene segment and a human IGHG1*01 CH3 gene segment.
 81. The methodof claim 1, wherein said human gamma-2 constant region of said mousecomprises a human IGHG2*01 CH2 gene segment and a human IGHG2*01 CH3gene segment.
 82. The method of claim 1, wherein said gene locus of saidmouse comprises a human gamma-3 constant region and/or a human gamma-4constant region.
 83. The method of claim 82, wherein said human gamma-3constant region of said mouse comprises a human IGHG3*04 CH2 genesegment and a human IGHG3*04 CH3 gene segment.
 84. The method of claim1, wherein said gene locus of said mouse comprises a human alphaconstant region.
 85. The method of claim 1, wherein said gene locus ofsaid mouse comprises a human delta constant region.
 86. The method ofclaim 1, wherein said gene locus of said mouse comprises a human epsilonconstant region.
 87. The method of claim 1, wherein said gamma-1 and/orgamma-2 constant region of said mouse excludes a CH1 gene segment. 88.The method of claim 1, wherein said gene locus of said mouse comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence consisting of the sequence from human gamma-3 to(and including) human gamma-4 constant regions; (b) a nucleotidesequence consisting of the sequence from human gamma-3 to (andincluding) human gamma-2 constant regions; (c) a nucleotide sequenceconsisting of the sequence from human gamma-3 to (and including) humangamma-1 constant regions; (d) a nucleotide sequence consisting of thesequence from human gamma-1 to (and including) human gamma-4 constantregions; (e) a nucleotide sequence consisting of the sequence from humangamma-1 to (and including) human gamma-2 constant regions; and (f) anucleotide sequence consisting of the sequence from human gamma-2 to(and including) human gamma-4 constant regions.
 89. The method of claim1, wherein said gene locus of said mouse comprises a nucleotide sequenceselected from the group consisting of: (i) a nucleotide sequenceconsisting of the sequence from human delta to (and including) humangamma-4 constant regions; (ii) a nucleotide sequence consisting of thesequence from human delta to (and including) human gamma-2 constantregions; (iii) a nucleotide sequence consisting of the sequence fromhuman delta to (and including) human alpha-1 constant regions; (iv) anucleotide sequence consisting of the sequence from human delta to (andincluding) human gamma-1 constant regions; and (v) a nucleotide sequenceconsisting of the sequence from human delta to (and including) humangamma-3 constant regions.
 90. The method of claim 25, wherein said humangamma-1 constant region of said mouse comprises a human IGHG1*01 CH2gene segment and a human IGHG1*01 CH3 gene segment.
 91. The method ofclaim 25, wherein said human gamma-2 constant region of said mousecomprises a human IGHG2*01 CH2 gene segment and a human IGHG2*01 CH3gene segment.
 92. The method of claim 25, wherein said gene locus ofsaid mouse comprises a human gamma-3 constant region and/or a humangamma-4 constant region.
 93. The method of claim 92, wherein said humangamma-3 constant region comprises a human IGHG3*04 CH2 gene segment anda human IGHG3*04 CH3 gene segment.
 94. The method of claim 25, whereinsaid gene locus of said mouse comprises a human alpha constant region.95. The method of claim 25, wherein said gene locus of said mousecomprises a human delta constant region.
 96. The method of claim 25,wherein said gene locus of said mouse comprises a human epsilon constantregion.
 97. The method of claim 25, wherein said gamma-1 and/or gamma-2constant region of said mouse excludes a CH1 gene segment.
 98. Themethod of claim 25, wherein said gene locus of said mouse comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence consisting of the sequence from human gamma-3 to(and including) human gamma-4 constant regions; (b) a nucleotidesequence consisting of the sequence from human gamma-3 to (andincluding) human gamma-2 constant regions; (c) a nucleotide sequenceconsisting of the sequence from human gamma-3 to (and including) humangamma-1 constant regions; (d) a nucleotide sequence consisting of thesequence from human gamma-1 to (and including) human gamma-4 constantregions; (e) a nucleotide sequence consisting of the sequence from humangamma-1 to (and including) human gamma-2 constant regions; and (f) anucleotide sequence consisting of the sequence from human gamma-2 to(and including) human gamma-4 constant regions.
 99. The method of claim25, wherein said gene locus of said mouse comprises a nucleotidesequence selected from the group consisting of (i) a nucleotide sequenceconsisting of the sequence from human delta to (and including) humangamma-4 constant regions; (ii) a nucleotide sequence consisting of thesequence from human delta to (and including) human gamma-2 constantregions; (iii) a nucleotide sequence consisting of the sequence fromhuman delta to (and including) human alpha-1 constant regions; (iv) anucleotide sequence consisting of the sequence from human delta to (andincluding) human gamma-1 constant regions; and (v) a nucleotide sequenceconsisting of the sequence from human delta to (and including) humangamma-3 constant regions.
 100. The method of claim 48, wherein saidhuman gamma-1 constant region of said mouse comprises a human IGHG1*01CH2 gene segment and a human IGHG1*01 CH3 gene segment.
 101. The methodof claim 48, wherein said human gamma-2 constant region of said mousecomprises a human IGHG2*01 CH2 gene segment and a human IGHG2*01 CH3gene segment.
 102. The method of claim 48, wherein said gene locus ofsaid mouse comprises a human gamma-3 constant region and/or a humangamma-4 constant region.
 103. The method of claim 102, wherein saidhuman gamma-3 constant region comprises a human IGHG3*04 CH2 genesegment and a human IGHG3*04 CH3 gene segment.
 104. The method of claim48, wherein said gene locus of said mouse comprises a human alphaconstant region.
 105. The method of claim 48, wherein said gene locus ofsaid mouse comprises a human delta constant region.
 106. The method ofclaim 48, wherein said gene locus of said mouse comprises a humanepsilon constant region.
 107. The method of claim 48, wherein saidgamma-1 and/or gamma-2 constant region of said mouse excludes a CH1 genesegment.
 108. The method of claim 48, wherein said gene locus of saidmouse comprises a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence consisting of the sequence from humangamma-3 to (and including) human gamma-4 constant regions; (b) anucleotide sequence consisting of the sequence from human gamma-3 to(and including) human gamma-2 constant regions; (c) a nucleotidesequence consisting of the sequence from human gamma-3 to (andincluding) human gamma-1 constant regions; (d) a nucleotide sequenceconsisting of the sequence from human gamma-1 to (and including) humangamma-4 constant regions; (e) a nucleotide sequence consisting of thesequence from human gamma-1 to (and including) human gamma-2 constantregions; and (f) a nucleotide sequence consisting of the sequence fromhuman gamma-2 to (and including) human gamma-4 constant regions. 109.The method of claim 48, wherein said gene locus of said mouse comprisesa nucleotide sequence selected from the group consisting of (i) anucleotide sequence consisting of the sequence from human delta to (andincluding) human gamma-4 constant regions; (ii) a nucleotide sequenceconsisting of the sequence from human delta to (and including) humangamma-2 constant regions; (iii) a nucleotide sequence consisting of thesequence from human delta to (and including) human alpha-1 constantregions; (iv) a nucleotide sequence consisting of the sequence fromhuman delta to (and including) human gamma-1 constant regions; and (v) anucleotide sequence consisting of the sequence from human delta to (andincluding) human gamma-3 constant regions.
 110. A method of obtaining anantigen-specific antibody or antigen binding fragment thereof, saidantibody comprising a human immunoglobulin heavy (IgH) chain comprisinga human IgH chain variable region and a human IgH chain constant region,said antigen binding fragment comprising said human IgH chain variableregion, the method comprising: expressing said antibody or antigenbinding fragment thereof from a cell comprising nucleic acid encodingsaid human IgH chain variable region of said antibody, wherein saidhuman IgH chain variable region is of a mouse whose genome comprises agene locus for expression of antibody heavy chains, said locuscomprising: (a) an unrearranged human heavy chain variable regioncomprising human heavy chain variable region gene segments comprising aplurality of unrearranged human variable gene segments (VH), one or moreunrearranged human D gene segments (D) and one or more human JH genesegments (JH) for expression of a plurality of human heavy chainvariable domains; and (b) a heavy chain constant region comprising thehuman gamma-1 constant region, wherein said human gamma constant regioncomprises CH2, CH3, M1 and M2 gene segments, said (a) and (b) encodinggamma-1 antibody heavy chains comprising (i) gamma-1 heavy chainconstant domain and (ii) human heavy chain variable domains; whereinsaid unrearranged human heavy chain variable region is upstream of andoperably linked to said human gamma-1 heavy chain constant region andcomprised by an IgH locus, wherein said human CH2 and human CH3 genesegments physically replace the respective CH2 and CH3 gene segments ofendogenous mouse gamma-1 heavy chain constant region DNA, such that saidhuman heavy chain variable region gene segments are able to recombinefor expression of a gamma-1 antibody heavy chain comprising a humangamma-1 heavy chain antibody Fc region comprising a human CH2 and ahuman CH3 and a human heavy chain variable domain.
 111. A method ofobtaining an antigen-specific antibody or antigen binding fragmentthereof, said antibody comprising a human immunoglobulin heavy (IgH)chain comprising a human IgH chain variable region and a human IgH chainconstant region, said antigen binding fragment comprising said human IgHchain variable region, the method comprising: expressing said antibodyor antigen binding fragment thereof from a cell comprising nucleic acidencoding said human IgH chain variable region of said antibody, whereinsaid human IgH chain variable region is of a mouse whose genomecomprises a gene locus for expression of antibody heavy chains, whereinsaid mouse has been produced comprising: (i) targeted insertion of saidhuman IgH chain variable region and said human IgH chain constant regionDNA at the endogenous IgH locus of an ES cell of a bacterial artificialchromosomes (BAC), wherein said BAC comprises the entire sequence ofhuman constant region DNA, (ii) implanting the ES cell produced in (i)into a donor blastocyst, (iii) implanting the blastocyst produced in(iii) into a foster mother; (iv) selecting mice with germlinetransmission of said human constant region DNA thereby producing micehaving said human constant region DNA integrated at the endogenous mouseIgH locus, (a) wherein said human IgH chain variable region comprises anunrearranged human heavy chain variable region comprising human heavychain variable region gene segments comprising a plurality ofunrearranged human variable gene segments (VH), one or more unrearrangedhuman D gene segments (D) and one or more human JH gene segments (JH)for expression of a plurality of human heavy chain variable domains; and(b) wherein said human IgH chain constant region comprises a heavy chainconstant region comprising the human gamma constant region, wherein saidhuman gamma constant region comprises CH2, CH3, M1 and M2 gene segments,said (a) and (b) encoding gamma antibody heavy chains comprising (i)gamma heavy chain constant domain and (ii) human heavy chain variabledomains; wherein said unrearranged human heavy chain variable region isupstream of and operably linked to said human gamma heavy chain constantregion and comprised by an IgH locus, wherein said human CH2 and humanCH3 gene segments are in place of the respective CH2 and CH3 genesegments of endogenous mouse gamma heavy chain constant region DNA, suchthat said human heavy chain variable region gene segments are able torecombine for expression of a gamma antibody heavy chain comprising ahuman gamma heavy chain antibody Fc region comprising a human CH2 and ahuman CH3 and a human heavy chain variable domain.